Aptamers Specific for TLR-4 and Uses Thereof

ABSTRACT

The invention relates to a nucleic acid aptamer with the capability of binding specifically to a and inhibiting TLR-4, to a complex comprising said aptamer and a functional group, as well as to pharmaceutical compositions thereof. The invention also relates to uses and methods for detecting TLR-4 and to uses and methods for inhibiting TLR-4. Finally, the invention also relates to an aptamer for use in manufacturing a drug for the treatment of a pathology characterized by an increase in expression of TLR4 and/or an increase in activation of TLR-4.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.17/022,984, with a filing date of Sep. 16, 2020, which issued as U.S.Pat. No. 11,591,603, on Feb. 28, 2023, which is a continuation of Ser.No. 16/220,726, with a filing date of Dec. 14, 2018, which issued asU.S. Pat. No. 10,808,252, on Oct. 20, 2020, which is a continuation ofSer. No. 15/322,021 with a 371(c) date of Dec. 23, 2016, which issued asU.S. Pat. No. 10,196,642, on Feb. 5, 2019, which is the U.S. nationalphase of International Application No. PCT/EP2015/064277, filed Jun. 24,2015, which claims the benefit of Spanish Patent Application No.P201430955, filed Jun. 24, 2014, all of which are herein incorporated byreference in their entireties.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:3951_0010004_SequenceListing_ST26.xml; Size: 9,036 bytes; and Date ofCreation: May 9, 2023) filed with the application is incorporated hereinby reference in its entirety.

FIELD OF THE INVENTION

The present invention provides nucleic acid aptamers with the capabilityof binding specifically to and inhibiting TLR-4 and uses thereof.

BACKGROUND OF THE INVENTION

It is known today that the Central Nervous System (CNS) responds both tobacterial infections and to brain damage, with a very well organizedinnate immune reaction. The innate immune system can recognize highlyconserved molecular patterns through, inter alia, toll-like receptors(TLR).

TLR4 was the first TLR characterized in mammals. Exogenous ligands havebeen described for this TLR, such as lipopolysaccharide (LPS) ofgram-negative bacteria, lipoteichoic acid (LTA) of gram-positivebacteria, or protein F of syncytial respiratory virus. Furthermore, themost important endogenous ligands are HMBG1, HSP-60 of an endogenousorigin or derived from Chlamydia pneumoniae, HPS-70, fibronectin,fibrinogen, hyaluronic acid, etc., all derived from tissue damage, celldamage and/or from the host's vessels. TLR4 is involved in a largenumber of highly prevalent pathologies, such as stroke orcerebrovascular disease, acute myocardial infarction, sepsis,atherosclerosis, multiple sclerosis, rheumatoid arthritis, a retinaldegenerative disease, and drug addiction, inter alia.

The involvement of innate immunity and, in particular, of TLRs inmultiple pathologies has sparked growing interest in the development ofagonists and antagonists of these receptors. Agonists have thereforebeen developed for the possible treatment of cancer, allergic diseases,infections, and as vaccine coadyuvants. In addition, TLR antagonists arebeing studied in sepsis, in atherosclerosis, in chronic pain and incolitis; in fact there are several antagonists, eritoran (phase III),ibudilast (Av411; phase II) and NI-0101 antibodies (pre-clinical phase),which are being studied in these pathologies.

Patent document WO 2006/138681 describes a method for inhibitingintrahepatic activated T-cell deletion by means of administering a TLR-4inhibitor, among which TLR-4-specific aptamers are mentioned.

Roger and others (Roger et al., 2009, Proc Natl Acad Sci USA106:2348-52) describe antibodies specific for the extracellular domainof TLR4. These antibodies provide protection against lethal sepsis ofgram-negative bacteria in mice. The therapeutic usefulness of theseanti-TLR4 antibodies is also suggested given that treatment is effectivewhen the antibodies are administered up to 4 h after exposure to anendotoxin and up to 13 h after the onset of infection due to Escherichiacoli.

Therefore, there is a need in the art for new molecules with thecapability of binding specifically to and inhibiting TLR-4 and that areuseful as therapeutic agents.

BRIEF DESCRIPTION OF THE INVENTION

In a first aspect, the present invention relates to a nucleic acidaptamer with the capability of binding specifically to and inhibitingTLR-4 and comprising a sequence selected from the group consisting ofSEQ ID NO: 1 and SEQ ID NO: 2 or a functionally equivalent variantthereof.

In another aspect, the present invention relates to a complex comprisingthe aptamer of the invention and a functional group.

In another aspect, the present invention relates to the use of theaptamer of the invention or of the complex of the invention fordetecting TLR-4.

In another aspect, the present invention relates to the in vitro use ofthe aptamer of the invention or of the complex of the invention forinhibiting TLR-4.

In another aspect, the present invention relates to an in vitro methodfor the detection of TLR-4 in a sample comprising

-   -   i) contacting said sample with an aptamer according to the        invention, or a complex according to the invention,    -   ii) separating the aptamer or complex not bound to TLR-4, and    -   iii) detecting the presence of the aptamer or complex bound to        the TLR-4 present in the sample.

In another aspect, the present invention relates to an in vitro methodfor inhibiting TLR-4 in a sample, which comprises contacting a samplecomprising TLR-4 with an aptamer according to the invention, or acomplex according to the invention, in conditions suitable forinhibiting TLR-4.

In another aspect, the present invention relates to an aptamer of theinvention for use in the treatment of a pathology characterized by anincrease in expression of TLR4 and/or an increase in activation ofTLR-4.

In another aspect, the present invention relates to a pharmaceuticalcomposition comprising at least one aptamer according to the inventionor at least one complex according to the invention, optionally incombination with one or more pharmaceutically acceptable carriers,excipients or solvents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . Recognition of the TLR-4 protein by the aptamers selected bymeans of ELONA. The human recombinant TLR-4 protein (6×HIS-TLR-4) wascultured at a concentration of 100 ng/well in 96-well microtiter platesand incubated at 4° C. for 16 h. Subsequently, 20 pmol of each of theaptamers labeled with digoxigenin were added to each well and the platewas incubated for 1 h at 37° C. Finally, the plate was incubated withperoxidase-conjugated anti-digoxigenin antibodies and developed usingABTS. An anti-Li H2A DNA aptamer was used as a positive control (Martinet al., 2013, PLoS ONE 8: e78886). All the experiments were performed intriplicate.

FIG. 2 . Secondary structures of the aptamers TLRApt #1R (SEQ ID NO: 3)and TLRApt #4F (SEQ ID NO: 4) predicted using the mFold program. Theguanines that could be part of predicted G-quadruplex structures areshown in the boxes with the QGRS Mapper program.

FIG. 3 . Binding of aptamers TLRApt #1R (SEQ ID NO: 3) and TLRApt #4F(SEQ ID NO: 4) to recombinant hTLR-4 (A) and to the TLR-4 proteinexpressed in cells (B). All the experiments were done in triplicate.

FIG. 4 . Antagonist effect of aptamers TLRApt #1R (SEQ ID NO: 3) andTLRApt #4F (SEQ ID NO: 4) on HEK-Blue hTLR4 cells and antagonistLPS-RS-UP (2 ng/μl; 20 ng) as control. Aptamers were applied at endconcentrations of 0.2, 2, 20 and nM or the antagonist control LPS-RS-UP(2 ng/μl; 20 ng). The agonist LPS-EK-UP (0.02 ng/μl) (A) or lysates fromHEK293 cells (Damage-associated molecular pattern; DAMP) (B) were usedas agonist control and secreted alkaline phosphatase (SEAP) activity wasmeasured after 24 h using QUANTI-Blue™ substrate at 630 nm. Data areexpressed as the percentage of SEAP activity relative to the controlcells. All the experiments were done in triplicate and average of 7-9different experiments is shown in the figure. Statistical significance(*P<0.05, **P<0.01 and ***P<0.001).

FIG. 5 . Effect of aptamers TLRApt #4F-T (SEQ ID NO: 2) and TLRApt #4F(SEQ ID NO: 4) on macrophages stimulated in the presence of 500 ng/mlLPS. Nitrites release was studied by the Griess reaction at 24 h aptameraddiction. Samples were assayed in duplicate. The differences wereanalyzed by one-way ANOVA followed by Bonferroni test. The result is theaverage of three experiments tested in duplicate. Statisticalsignificance (***P<0.001). Keys: 38x (AG) is the oligonucleotide ofsequence SEQ ID NO: 7, which is a nonspecific sequence that is not ableto adopt any secondary structure; lnh: hispanolone derivative compound11 (Girón et al., 2008, Toxicol Appl Pharmacol 228:179-89).

FIG. 6 . Secondary structures of aptamers TLRApt #1R-T (SEQ ID NO: 1)and TLRApt #4F-T (SEQ ID NO: 2) predicted using the mFold program. Theguanines that could be part of predicted G-quadruplex structures areshown in the boxes with the QGRS Mapper program.

FIG. 7 . Effect of the intraperitoneal injection of 1 nmol of aptamersTLRApt #1R-T (SEQ ID NO: 1), TLRApt #4F-T (SEQ ID NO: 2) and TLRApt #4F(SEQ ID NO: 4) or 38x(AG) (SEQ ID NO: 7) or vehicle (PBS+1 mM Mg²⁺) inthe reduction of the infarcted area in animals used in experiments.Adult male mice C57BL/10ScSn (WT; normal) and C57BL/10ScNJ (KO, lackingfunctional TLR4), were subjected to induction of a focal cerebralischemia by means of occlusion of the middle cerebral artery vialigature. The mice were anesthetized with isoflurane and 24 hours afterMCAO, the size of the infarction was evaluated by MRI. The imageshighlighted in T2 (T2WI) have been acquired in a BIOSPEC BMT 47/40operating at 4.7 T (Bruker-Medical, Ettlingen, Germany; MRI Unit,Instituto Pluridisciplinar, UCM) and the damaged area is quantified bymeans of Image J 1.41 (NIH, Bethesda, Wash.). Statistical significance(*P<0.05). Keys: 1RT, TLRApt #1R-T; 4FT, TLRApt #4F-T; 4F, TLRApt #4F.

FIG. 8 : Dose-response curve. Effect of the intraperitoneal injection ofdifferent amounts of aptamers TLRApt #4F-T (SEQ ID NO: 2) and TLRApt #4F(SEQ ID NO: 4) or vehicle (PBS+1 mM Mg2+) in the reduction of theinfarcted area in animals used in experiments. Adult male miceC57BL/10ScSn (WT; normal were subjected to induction of a focal cerebralischemia by means of occlusion of the middle cerebral artery vialigature. The mice were anesthetized with isoflurane and 24 hours afterMCAO, the size of the infarction was evaluated by MRI. The imageshighlighted in T2 (T2WI) have been acquired in a BIOSPEC BMT 47/40operating at 4.7 T (Bruker-Medical, Ettlingen, Germany; MRI Unit,Instituto Pluridisciplinar, UCM) and the damaged area is quantified bymeans of Image J 1.41 (NIH, Bethesda, Wash.). Statistical significance(*P<0.05).

FIG. 9 : Flow cytometry assays. (A) Human HEK293 (left panel) and293-hTLR4A (right panel) cell lines were incubated with 20 nM Alexafluor 488-labeled aptamers for 30 min at room temperature. The cellswere washed with 2 ml of PBS and resuspended in 1 mL PBS for analysis.In each figure, the ordinate represents the frequency of events (or cellnumber) while the abscissa indicates the fluorescence intensity (FL1).Black area, autofluorescence; black line, aptamer TLRApt #1R-T (SEQ IDNO: 31); grey line, aptamer TLRApt #4F-T (SEQ ID NO: 42). (B) The human293-hTLR4A cell line is activated with LPS-EK-UP and then incubated 30min with 20 nM of Alexa Fluor 488-labeled aptamers for 30 min at roomtemperature. The cells are washed with 2 ml PBS and resuspended in 1 mLPBS for analysis. In each figure, the ordinate represents the frequencyof events (or cell number) while the abscissa indicates the fluorescenceintensity (FL1). Black area, autofluorescence; black line, aptamerTLRApt #1R-T (SEQ ID NO: 31); grey line, aptamer TLRApt #4F-T (SEQ IDNO: 42).

FIG. 10 : Analysis of the half-life of the aptamers in vitro. Threehundred ng of folded aptamers were incubated with 2 units of AExonuclease or DNAse I for several periods of time at 37° C. Afterward,samples were solved on a 3% agarose gel and bands visualized by GelRedand quantified using Image Studio Digits V3.1 software.

DETAILED DESCRIPTION OF THE INVENTION

The authors of the present invention have selected and characterized twomolecules which, due to their sequences, can be three-dimensionallystructured in certain pH, temperature and saline concentrationconditions, giving them the capability to recognize specifically theTLR-4 protein and modulate its activity. These molecules can inhibitcellular response mediated by receptor TLR-4 in vivo and can reduce thesize of brain infarction in animal models of ischemic stroke, givingthem a potential therapeutic role.

Aptamer Specific for TLR-4

In a first aspect, the present invention relates to a nucleic acidaptamer with the capability of binding specifically to and inhibitingTLR-4, hereinafter referred to as the “aptamer of the invention”, andcomprising a sequence selected from the group consisting of SEQ ID NO: 1(CCGGCACGGGACAAGGCGCGGGACGGCGTAGATCAGGTCGACACC) and SEQ ID NO: 2(GGTGTGCCAATAAACCATATCGCCGCGTTAGCATGTACTCGGT TGGCCCTAAATACGAG), or afunctionally equivalent variant thereof.

The term “aptamer”, in the context of the present invention, refers tosingle-stranded nucleic acid chains adopting a specific tertiarystructure that allows them to bind to molecular targets with highspecificity and affinity, comparable to that of monoclonal antibodies,through interactions other than conventional Watson-Crick base pairing.

The term “nucleic acid”, in the context of the present invention, refersto any type of nucleic acid, such as DNA and RNA, and to variantsthereof, such as peptide nucleic acid (PNA), locked nucleic acid (LNA),as well as combinations thereof, modifications thereof, includingmodified nucleotides, etc. The terms “nucleic acid” and“oligonucleotide” and “polynucleotide” are used interchangeably in thecontext of the present invention. Nucleic acids can be purified fromnatural sources, produced using recombinant expression systems and,optionally, purified, chemically synthesized, etc. When appropriate, forexample, in the case of chemically synthesized molecules, the nucleicacids can comprise nucleoside analogues such as analogues havingchemically modified bases or sugars, modifications of the backbone, etc.A nucleic acid sequence is represented in 5′-3′ direction unlessindicated otherwise.

The term “TLR-4”, in the context of the present invention, refers tomembrane receptor toll-like receptor 4. Receptor TLR-4 can also bereferred to as ARMD10, CD284, TLR4 or hTOLL. In humans, receptor TLR-4was registered in GenBank under accession number 000206.2 on 27th May,2014, and it is encoded by the TLR4 gene. It is made up of 839 aminoacids, of which residues 1-23 constitute the signal sequence, residues24-631 constitute the extracellular domain, residues 632-652 constitutethe transmembrane domain, and residues 653-839 constitute thecytoplasmic domain.

In a particular embodiment, the aptamer can bind specifically to theextracellular domain of TLR-4 (amino acids 24-631).

The present invention contemplates an aptamer comprising a sequenceselected from the group consisting of SEQ ID NO: 1(CCGGCACGGGACAAGGCGCGGGACGGCGTAGATCAGGTCGACACC) and SEQ ID NO: 2(GGTGTGCCAATAAACCATATCGCCGCGTTAGCATGTACTCGGT TGGCCCTAAATACGAG) or afunctionally equivalent variant thereof.

The present invention also contemplates aptamers of the invention thatare made up of nucleic acids such as DNA and RNA, as well as of nucleicacid variants and analogues and combinations thereof, modificationsthereof, including, without limitation, modified nucleic acid backbones,substitution bonds, modified nucleotides, and ribose or deoxyriboseanalogues, modified nucleotides, etc., with a capability of bindingspecifically to and inhibiting TLR-4 of at least 50%, at least 60%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or at least 100% ofthe capability of specifically binding to and inhibiting TLR-4 of theaptamer of sequence SEQ ID NO: 1 or SEQ ID NO: 2. Non-limiting examplesof nucleic acid variants and analogues include, without limitation, PNA,LNA and TNA.

The term “nucleic acid variant” or “nucleic acid analogue”, in thecontext of the present invention, refers to nucleic acid variants andanalogues including, without limitation, modified nucleic acidbackbones, substitution bonds, modified nucleotides, and ribose ordeoxyribose analogues. For example, nucleic acid variants according tothe present invention can comprise structures with analogue syntheticbackbones of the typical phosphodiester backbone. These include, withoutlimitation, phosphorothioate, phosphorodithioate, methylphosphonate,phosphoramidate, alkyl phosphotriester, sulfamate, 3′-thioacetal,methylene(methylimino), 3′-N-carbamate, morpholine carbamate and peptidenucleic acids (PNA), methylphosphonate bonds or alternatingmethylphosphonate and phosphodiester and benzylphosphonate.

Nucleic acid variants can also contain one or more “substitution” bonds,as is generally understood in the art. Some of these substitution bondsare apolar and contribute to providing the aptamer with a capability ofspreading through the membranes. These “substitution” bonds are hereindefined as conventional alternative bonds, such as phosphorothioate orphosphoramidate, and are synthesized as described in the commonlyavailable literature. Alternative binding groups include, in anon-limiting manner, embodiments in which a moiety of formula P(O)S,(“thioate”), P(S)S (“dithioate”), P(O)NR′₂, P(O)R′, P(O)OR⁶, CO, orCONR′₂, wherein R′ is H (or a salt) or an alkyl group of 1-12 carbonatoms and R⁶ is an alkyl group of 1-9 carbon atoms, which binds toadjacent nucleotides through —S— or —O—. Dithioate bonds are describedin US patent application 248517. The present invention also contemplatesthe use of substitution bonds including internucleotide bonds not basedon phosphorus, such as 3′-thioformacetal, (—S—CH₂—O—), formacetal(—O—CH₂—O—) and 3′-amine internucleotide bonds (—NH—CH₂—CH₂—) describedin U.S. Pat. No. 690,786 and 763,130. One or more substitution bonds canbe used in the aptamers of the invention for the purpose of even furtherfacilitating binding to TLR-4 or for increasing the stability of theaptamers against nucleases, as well as for providing permeationcapability. Not all the bonds within the same aptamer have to beidentical, and the present invention therefore contemplates aptamerswith all identical bonds as well as aptamers with a variation in thecomposition of their bonds.

Likewise, nucleic acid variants according to the present invention canalso contain ribose or deoxyribose analogue forms which are well-knownin the art, including without limitation sugars substituted at 2′, suchas 2′-O-methyl-ribose, 2′-fluoro-ribose or 2′-azido-ribose, carbocyclicanalogues of sugars, α-anomeric sugars, epimeric sugars such asarabinose, xyloses or lyxoses, pyranose sugars, furanose sugars andsedoheptuloses. The nucleic acids can also contain threose nucleic acid(TNA, also referred to as alpha-threofuranosyl oligonucleotides) (see,for example, Schong et al., Science 2000, November 17, 290 (5495):1347-1351). In particular, the substitution in the position 2′ of theresidue of furanose is particularly important with respect to theimprovement in nuclease stability.

The term “nucleotide”, in the context of the present invention, refersto the monomers making up the nucleic acids. The nucleotides are formedby a pentose, a nitrogenous base and a phosphate group, and are bound bymeans of phosphodiester bonds. The nucleotides that are part of DNA andRNA differ in the pentose, this being deoxyribose and ribose,respectively. The nitrogenous bases, in turn, are divided into purinenitrogenous bases, which are adenine (A) and guanine (G), and intopyrimidine nitrogenous bases, which are thymine (T), cytosine (C) anduracil (U). Thymine only appears in DNA, whereas uracil only appears inRNA. The present invention contemplates the use of modified nucleotidesin the aptamer of the invention. The term “modified nucleotide”, in thecontext of the present invention, refers to known natural nucleotidesanalogues, with similar or improved binding properties. Analogue formsof purines and pyrimidines are well-known in the art and include,without limitation, aziridinylcytosine, 4-acetylcytosine,5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, inosine, N₆-isopentenyladenine,1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine,2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,5-methylcytosine, N₆-methyladenine, 7-methylguanine,5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,beta-D-mannosylkeosine, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid methylester, pseudouracil, keosine, 2-thiocytosine, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid, and2,6-diaminopurine. In addition to the preceding modified nucleotides,nucleotide residues lacking a purine or a pyrimidine also can beincluded in the present invention.

In addition to the preceding variants, the nucleic acid variantscomprised in the invention also include PNA, LNA and 5′-5′ or 3′-3′chains. The term “peptide nucleic acid” or “PNA”, in the context of thepresent invention, refers to an oligonucleotide the backbone of which ismade up of repetitive units of N-(2-aminoethyl)-glycine bound by peptidebonds, wherein the different nitrogenous bases are bound to the mainchain by a methylene bond (—CH₂—) and a carbonyl group (—(C═O)—). Theterm “locked nucleic acid” or “LNA”, in the context of the presentinvention, refers to a modified RNA nucleotide the ribose moiety ofwhich is modified with an additional bond connecting the oxygen at 2′with the carbon at 4′, locking the ribose in the 3′-endo conformation.The term “5′-5′ chain” or “3′-3′ chain”, in the context of the presentinvention, refers to oligonucleotides in which the nucleotide of the 3′or 5′ ends, respectively, is inverted.

As it is used herein, the term “functionally equivalent variant” refersto aptamers with sequences substantially similar to SEQ ID NO: 1 or SEQID NO: 2 maintaining the capability of specifically binding to andinhibiting TLR-4. A functionally equivalent variant of the aptamer ofthe invention can be a nucleic acid sequence derived from SEQ ID NO: 1or SEQ ID NO: 2 comprising the addition, substitution or modification ofone or more nucleotides. By way of illustration, functionally equivalentvariants of the aptamer of the invention include sequences comprisingthe addition of 1 nucleotide, 2 nucleotides, 3 nucleotides, 4nucleotides, 5 nucleotides, 10 nucleotides, 15 nucleotides, 20nucleotides, 25 nucleotides, 30 nucleotides, 35 nucleotides, 40nucleotides, 45 nucleotides, 50 nucleotides, 60 nucleotides, 70nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 150nucleotides, 200 nucleotides, at least nucleotides, at least 1000nucleotides or more at the 5′ end of the sequence SEQ ID NO: 1 or SEQ IDNO: 2, and/or comprising the addition of 1 nucleotide, 2 nucleotides, 3nucleotides, 4 nucleotides, 5 nucleotides, 10 nucleotides, 15nucleotides, 20 nucleotides, 25 nucleotides, 30 nucleotides, 35nucleotides, 40 nucleotides, 45 nucleotides, 50 nucleotides, 60nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100nucleotides, 150 nucleotides, 200 nucleotides, at least nucleotides, atleast 1000 nucleotides or more at the 3′ end of the sequence SEQ ID NO:1 or SEQ ID NO: 2, and maintaining a capability of specifically bindingto and inhibiting TLR-4 of at least 50%, at least 60%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or at least 100% of thecapability of specifically binding to and inhibiting TLR-4.

The present invention also includes aptamers comprising nucleotidesequences with a sequence identity of at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% with the sequences SEQ ID NO: 1 or SEQ ID NO:2 and maintaining a capability of specifically binding to and inhibitingTLR-4 of at least 50%, at least 60%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or at least 100% of the capability ofspecifically binding to and inhibiting TLR-4.

The terms “identity”, “identical” or “percent identity” in the contextof two or more nucleic acids, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides or amino acid residues that are the same, when compared andaligned (introducing gaps, if necessary) for maximum correspondence, notconsidering any conservative amino acid substitutions as part of thesequence identity. The percent identity can be measured using sequencecomparison software or algorithms or by visual inspection. Variousalgorithms and software are known in the art that can be used to obtainalignments of amino acid or nucleotide sequences. One such non-limitingexample of a sequence alignment algorithm is the algorithm described inKarlin et al., 1990, Proc. Natl. Acad. Sci., 87:2264-8, as modified inKarlin et al., 1993, Proc. Natl. Acad. Sci., 90:5873-7, and incorporatedinto the NBLAST and XBLAST programs (Altschul et al., 1991, NucleicAcids Res., 25:3389-402). In certain embodiments, Gapped BLAST can beused as described in Altschul et al., 1997, Nucleic Acids Res.25:3389-402. BLAST-2, WU-BLAST-2 (Altschul et al., 1996, Methods inEnzymology, 266:460-80), ALIGN, ALIGN-2 (Genentech, South San Francisco,Calif.) or Megalign (DNASTAR) are additional publicly available softwareprograms that can be used to align sequences. In certain embodiments,the percent identity between two nucleotide sequences is determinedusing the GAP program in GCG software (e.g., using a NWSgapdna.CMPmatrix and a gap weight of 40, 50, 60, 70, or 90 and a length weight of1, 2, 3, 4, 5, or 6). In certain alternative embodiments, the GAPprogram in the GCG software package, which incorporates the algorithm ofNeedleman and Wunsch (J. Mol. Biol. 48:444-53 (1970)) can be used todetermine the percent identity between two amino acid sequences (e.g.,using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5).Alternatively, in certain embodiments, the percent identity betweennucleotide or amino acid sequences is determined using the algorithm ofMyers and Miller (CABIOS, 4:11-7 (1989)). For example, the percentidentity can be determined using the ALIGN program (version 2.0) andusing a PAM120 with residue table, a gap length penalty of 12 and a gappenalty of 4. Appropriate parameters for maximal alignment by particularalignment software can be determined by one skilled in the art. Incertain embodiments, the default parameters of the alignment softwareare used. In certain embodiments, the percentage identity “X” of a firstamino acid sequence to a second sequence amino acid is calculated as100×(Y/Z), where Y is the number of amino acid residues scored asidentical matches in the alignment of the first and second sequences (asaligned by visual inspection or a particular sequence alignment program)and Z is the total number of residues in the second sequence. If thesecond sequence is longer than the first sequence, then the percentidentity may be determined only in the region of overlap between saidfirst and second sequences. In this case, the same formula as above canbe used but using as Z value the length of the region wherein the firstand second sequence overlaps, said region having a length which issubstantially the same as the length of the first sequence.

As a non-limiting example, whether any particular polynucleotide has acertain percentage sequence identity (e.g., is at least 80% identical,at least 85% identical, at least 90% identical, and in some embodiments,at least 95%, 96%, 97%, 98%, or 99% identical) to a reference sequencecan, in certain embodiments, be determined using the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). Bestfit uses the local homology algorithm of Smith andWaterman, Advances in Applied Mathematics 2:482-9 (1981), to find thebest segment of homology between two sequences. When using Bestfit orany other sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set such that thepercentage of identity is calculated over the full length of thereference nucleotide sequence and that gaps in homology of up to 5% ofthe total number of nucleotides in the reference sequence are allowed.

In some embodiments, two nucleic acids of the invention aresubstantially identical, meaning they have at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, and in some embodiments atleast 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity,when compared and aligned for maximum correspondence, as measured usinga sequence comparison algorithm or by visual inspection. Identity canexist over a region of the sequences that is at least about 10, about20, about 40-60 residues in length or any integral value therebetween,and can be over a longer region than 60-80 residues, for example, atleast about 90-100 residues, and in some embodiments, the sequences aresubstantially identical over the full length of the sequences beingcompared, such as the coding region of a nucleotide sequence forexample.

The term “specific binding” or “specific binding to TLR-4”, in thecontext of the present invention, refers to the non-covalent physicalassociation between two molecules, the aptamer of the invention andreceptor TLR-4. The binding between the aptamer of the invention andreceptor TLR-4 is considered specific if the binding strength betweenboth is at least 10 times, at least 15 times, at least 20 times, atleast times, at least 50 times, at least 75 times or at least 100 timesgreater than the binding strength between the aptamer of the inventionand an irrelevant molecule. The binding between the aptamer of theinvention and receptor TLR-4 is also considered specific if theequilibrium dissociation constant Kd is 10⁻³ M or less, 10⁻⁴ M or less,10⁻⁵ M or less, 10⁻⁶ M or less, 10⁻⁷ M or less, 10⁻⁸ M or less, 10⁻⁹ Mor less, 10⁻¹⁰ M or less, 10⁻¹¹ M or less, or 10⁻¹² M or less under theconditions used, for example, in physiological conditions, cell cultureconditions or conditions that allow cell survival.

The capability of the aptamer of the invention of binding specificallyto TLR-4 can be determined by means of a range of assays that areavailable in the art. Preferably, the capability of the aptamer of theinvention for the specific binding to TLR-4 is determined by means of invitro binding assays, such as the enzyme-linked oligonucleotide assay(ELONA), the enzyme-linked aptamer sorbent assay (ELASA), precipitationand quantitative PCR (qPCR), or by fluorescence techniques such asaptahistochemistry, aptacytochemistry, fluorescence microscopy or flowcytometry. Likewise, both the capability of specific binding to TLR-4and the affinity of the aptamer for TLR-4 can be determined bytechniques well-known by the person skilled in the art, such as gelmobility shift assay, surface plasmon resonance (SPR), kinetic capillaryelectrophoresis and fluorescence binding assay. Briefly, thefluorescence binding assay consists of the incubation of magnetic ballscoated with TLR-4 with different concentrations (for example, from 0 to100 nM) of the aptamer of the invention labeled (for example, withcarboxyfluorescein, FAM), and the subsequent elution and detection ofthe bound aptamers; the dissociation constants (Kd) are calculated bynon-linear fit analysis.

The term “inhibition of TLR-4”, in the context of the present invention,refers to the blocking or disminution of the activity of TLR-4, i.e.,the transduction of the receptor TLR-4-mediated signal. It is consideredthat the activity of TLR-4 is inhibited by an inhibitory agent orantagonist when its activity is at least 95%, at least 90%, at least85%, at least 80%, at least 75%, at least 70%, at least 65%, at least60%, at least 55%, at least 50%, at least 45%, at least 40%, at least35%, at least 30%, at least 25%, at least 20%, at least 15%, at least10%, at least 5%, at least 1%, or less of the activity of TLR-4 in thepresence of its natural agonist LPS.

The capability of the aptamer of the invention of inhibiting TLR-4 canbe determined by means of a range of assays that are available in theart. Preferably, the capability of inhibiting TLR-4 of the aptamer ofthe invention is determined by means of in vitro assays with cellsexpressing recombinant TLR-4 and a reporter gene, the expression ofwhich is associated with the activation of recombinant TLR-4. The personskilled in the art will recognize that there are multiple variants ofthis method, depending on the cell and the recombinant gene used. Anexample of this assay is included in the examples of the presentinvention (section on “Materials and methods” and Example 2). Otheravailable techniques include the determination of the levels ofinflammatory cytokines, such as IL-1, IL-8, TNF-alpha and IL-12,released by cells that express TLR-4.

In a particular embodiment, the aptamer of the invention consists ofbetween 30 and 200 nucleotides, preferably between 35 and 150nucleotides, more preferably between 40 and 100 nucleotides, even morepreferably between 45 and 80 nucleotides.

In another particular embodiment, the aptamer of the invention comprisesa sequence selected from the group consisting of SEQ ID NO: 3(GTTGCTCGTATTTAGGGCCACCGGCACGGGACAAGGCGCGGGACGGCGTAGATCAGGTCGACACCAGTCTTCATCCGC) and SEQ ID NO: 4 (GCGGATGAAGACTGGTGTGCCAATAAACCATATCGCCGCGTTAGCATGTACTCGGTTGGCCCTAAAT ACGAGCAAC). Thesequence SEQ ID NO: 3 is a functionally equivalent variant of SEQ ID NO:1 and the sequence SEQ ID NO: 4 is a functionally equivalent variant ofSEQ ID NO: 2.

In a particular embodiment, the nucleic acid is DNA. In anotherparticular embodiment, the nucleic acid is RNA. In another particularembodiment, the nucleic acid is PNA. In another particular embodiment,the nucleic acid is LNA. In another particular embodiment, the nucleicacid is TNA.

In another particular embodiment, the TLR-4 is a TLR-4 selected from thegroup formed by mouse, rat, rabbit, pig, cat, dog, horse, primate, andhuman TLR-4. In a preferred embodiment, the TLR-4 is a human TLR-4.

The production of the aptamer of the invention can be carried outfollowing conventional methods in the art. Non-limiting examples oftechniques for the production of aptamers include enzymatic techniques,such as transcription, recombinant expression systems and standard solidphase (or solution phase) chemical synthesis, all commerciallyavailable. When appropriate, for example, in the event that the aptamerof the invention comprises nucleic acid variants such as those describedabove, nucleotide analogues such as analogues having chemically modifiedbases or sugars, backbone modifications, etc., the aptamer of theinvention will be produced by means of chemical synthesis.Alternatively, expression will be the technique preferred for theproduction of aptamers when said aptamers have a length of 200nucleotides or more. The aptamers produced by or any of the precedingtechniques can optionally be purified by methods that are well known inthe art.

Complex of the Invention

As the person skilled in the art will appreciate, the features of thesmall size, stability and easy production of the aptamer of theinvention enable said aptamer to be presented bound to a secondmolecule. That is particularly advantageous when the second molecule isa functional group. The result of the binding of the aptamer of theinvention and a functional group is a complex presenting the combinationof functions of both, i.e., a complex with the capability ofspecifically binding to and inhibiting TLR-4 and with the activityassociated with the functional group.

Therefore, in another aspect, the present invention refers to a complex,hereinafter referred to as the “complex of the invention”, comprising anucleic acid aptamer with the capability of binding specifically to andinhibiting TLR-4, comprising a sequence selected from the groupconsisting of SEQ ID NO: 1 and SEQ ID NO: 2 or a functionally equivalentvariant thereof and a functional group.

The term “aptamer” has been described in detail in relation to theDefinitions and the Aptamer specific for TLR-4 (supra) and itsdefinitions and particularities apply likewise in the context of thecomplex of the invention.

The term “functional group”, in the context of the present invention,refers to compounds suitable for performing at least one function. Saidfunction includes, without limitation, the capability of bindingspecifically to TLR-4 or to other receptors TLR, the capability ofinhibiting TLR-4 or other receptors TLR, the capability of being bothdirectly and indirectly detectable, the capability of inducing celldeath, the capability of carrying a therapeutic payload, etc. As theperson skilled in the art will understand, a functional group can haveassociated therewith one or multiple functions. Non-limiting examples offunctional groups include detectable reagents and drugs. Thesefunctional groups act like imaging agents, drugs, etc.

Therefore, in a particular embodiment, the functional group is selectedfrom a detectable reagent, a drug and a nanoparticle.

In another particular embodiment, the functional group is a detectablereagent. The terms “detectable reagent”, “imaging agent” and “contrastagent” are used herein interchangeably and refer to a biocompatiblecompound, the use of which facilitates the differentiation of differentparts of the image, by increasing the “contrast” between those differentregions of the image. The term “contrast agents” thus encompasses agentsthat are used to enhance the quality of an image that may nonetheless begenerated in the absence of such an agent (as is the case, for instance,in MRI), as well as agents that are prerequisites for the generation ofan image (as is the case, for instance, in nuclear imaging). Suitablecontrast agent include, without limitation, contrast agents forradionuclide imaging, for computerized tomography (CT), for Ramanspectroscopy, for Magnetic resonance imaging (MRI) and for opticalimaging.

Detectable reagents for radionuclide imaging includeradiopharmaceuticals are commonly labeled with positron-emitters such as¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁸²Rb, ⁶²Cu, ⁶⁴Cu, and ⁶⁸Ga⁸⁶Y, ¹²⁴I, ²¹³Bi and²¹¹At. SPECT radiopharmaceuticals are commonly labelled with positronemitters such as ⁹⁴mTc, ²⁰¹Tl and ⁶⁷Ga. Radionuclide imaging modalities(positron emission tomography, (PET); single photon emission computedtomography (SPECT)) are diagnostic cross-sectional imaging techniquesthat map the location and concentration of radionuclide-labeledradiotracers. PET and SPECT can be used to localize and characterize aradionuclide by measuring metabolic activity. PET and SPECT provideinformation pertaining to information at the cellular level, such ascellular viability. In PET, a positron emitter is administered to thepatient, which can be monitored as the substance moves through the body.In certain embodiments of the invention, a complex according to theinvention is used for PET or SPECT imaging in vivo. Closely related toPET is single-photon emission computed tomography, or SPECT. The majordifference between the two is that instead of a positron-emittingsubstance, SPECT uses a radioactive tracer that emits low-energyphotons. Other non-limiting examples of radionuclides include gammaemission isotopes, such as ^(99m)Tc, ¹²³I and ¹¹¹In, which can be usedin radioscintigraphy using gamma cameras or computerized single photonemission tomography, as well as beta emitters, such as ¹³¹I, ⁹⁰Y,^(99m)Tc, ¹⁷⁷Lu and ⁶⁷Cu”. The person skilled in the art will understandthat the radionuclides may also be used for therapeutic purposes.

Detectable reagents for CT imaging include, for example, iodinated orbrominated contrast media. Examples of these agents include iothalamate,iohexyl, diatrizoate, iopamidol, ethiodol and iopanoate. Gadoliniumagents have also been reported to be of use as a CT contrast agent. Forexample, gadopentate agents have been used as a CT contrast agent.Computerized tomography (CT) is contemplated as an imaging modality inthe context of the present invention. By taking a series of X-rays,sometimes more than a thousand, from various angles and then combiningthem with a computer, CT made it possible to build up athree-dimensional image of any part of the body. A computer isprogrammed to display two-dimensional slices from any angle and at anydepth. In CT, intravenous injection of a radiopaque contrast agent suchas those described herein can assist in the identification anddelineation of soft tissue masses when initial CT scans are notdiagnostic.

Detectable reagents for optical imaging include, for example,fluorescein, a fluorescein derivative, indocyanine green, Oregon green,a derivative of Oregon green derivative, rhodamine green, a derivativeof rhodamine green, an eosin, an erythrosin, Texas red, a derivative ofTexas red, malachite green, nanogold sulfosuccinimidyl ester, cascadeblue, a coumarin derivative, a naphthalene, a pyridyloxazole derivative,cascade yellow dye, dapoxyl dye, and various other fluorescentcompounds, such as Cy3, Cy2, Cy5, the Alexa Fluor® fluorescent labelfamily (Molecular Probes, Inc.), carboxyfluorescein (FAM) andfluorescein isothiocyanate (FITC).

In another preferred embodiment, the detectable reagent is a protein.The term “protein”, in the context of the present invention, refers tomacromolecules consisting of one or more amino acid chains. The proteinsare responsible for carrying out a diverse group of cell functions basedon their ability to specifically bind other molecules. The proteins canbe bound to other proteins as well as to small substrate molecules.Non-limiting examples of proteins suitable for the purposes of thepresent invention include, without limitation, enzymes, fluorescentproteins, luminescent proteins and antigens.

In an even more preferred embodiment, the protein is an enzyme. The term“enzyme”, in the context of the present invention, refers to a proteinworking as a highly selective catalyst, accelerating both the speed andthe specificity of the metabolic reaction for which it is specific.Non-limiting examples of enzymes suitable for the invention include,without limitation, horseradish peroxidase (HRP) and alkalinephosphatase. As the person skilled in the art will understand, theenzymes suitable for use in the present invention are indirectlydetectable as a result of their capability of catalyzing modifying asubstrate in a compound detectable by colorimetry, chemiluminescence orfluorimetry. Examples of suitable substrates include, withoutlimitation, p-Nitrophenyl phosphate (PNPP),2,2′-azinobis[3-ethylbenzothiazolin-6-sulfonic acid] (ABTS),o-phenylenediamine (OPD), and 3,3′,5,5′-tetramethylbenzidine (TMB).

Bioluminescent proteins or photoproteins are a particular case ofoxidative enzymes capable of carrying out a chemical reaction of theirspecific prosthetic groups, resulting in light emission withoutrequiring prior excitation. Non-limiting examples of bioluminescentproteins include firefly luciferase, Renilla luciferase and aequorin.

In another even more preferred embodiment, the protein is a fluorescentprotein. The term “fluorescent protein”, in the context of the presentinvention, refers to a protein with the capability of emitting lightwhen it is excited at a wavelength suitable for excitation. Non-limitingexamples of fluorescent proteins that can be used in the complex of theinvention include, without limitation, GFP, GFPuv, BFP, CFP, YFP, EBFP2,mCerulean, mCerulean3, mVenus, mTurquoise, T-Sapphire, citrine, amFP486,zFP506, zFP538, drFP, DsRed, mCherry, dTomate, mTFP1, TagRFP-T, mKO2,mRuby, mKate, mAmetrine, REACh, R-phycoerythrin (R-PE) andAllophycocyanin (APC).

In another even more preferred embodiment, the protein is a luminescentprotein. The term “luminescent protein”, in the context of the presentinvention, refers to a protein capable of emitting light when it isexcited at a wavelength suitable for excitation. Non-limiting examplesof fluorescent proteins that can be used in the complex of the inventioninclude, without limitation, the proteins included in Table 1, togetherwith their corresponding excitation and emission wavelengths.

In another even more preferred embodiment, the protein is an antigen.The term “antigen”, in the context of the present invention, refers to amolecule that induces an immune response in the body. Therefore, anantigen can be used for generating an antibody that recognizes it andbinds specifically to it. Non-limiting examples of antigens include,inter alia, tumor antigens, such as the carcinoembryonic antigen (CEA),HER2, prostate specific antigen (PSA) and tissue plasminogen activatorand its recombinant variants, such as Activase®, as well as bacterialantigens, allergens, etc. As the person skilled in the art willunderstand, the antigens suitable for use in the present invention areindirectly detectable as a result of their capability of beingspecifically recognized by an antibody.

In another preferred embodiment, the detectable reagent is a haptene.The term “haptene”, in the context of the present invention, refers to agroup of chemical compounds having a small molecular size (<10,000 Da)which are antigenic but unable to induce by themselves an specificimmune reaction. The chemical coupling of a haptene to a largeimmunogenic protein, called carrier, generates an haptene-immunogeniccarrier conjugate which is able to induce a specific immune reaction.Non-limiting examples of vitamins include biotin (vitamin B7),digoxigenin, dinitrophenol (DNP) and nitro-iodophenol (NIP). In a morepreferred embodiment, the vitamin is biotin. The term “biotin”, in thecontext of the present invention, refers to a water- and alcohol-solubleheat-stable vitamin, also referred to as vitamin H and vitamin B7,characterized by specifically binding to avidin with the highestaffinity described to date of Kd=10⁻¹⁵ M. As the person skilled in theart will understand, biotin is indirectly detectable as a result of itscapability of being specifically recognized by avidin or variantsthereof, such as streptavidin and neutravidin.

In another particular embodiment, the functional group is a drug. Theterm “drug”, in the context of the present invention, refers to achemical substance used in the treatment, cure or prevention of adisease or condition, such as a pathology characterized by an increasein expression of TLR-4 and/or an increase in activation of TLR-4. Theterm “pathology characterized by an increase in expression of TLR-4and/or an increase in activation of TLR-4” is described in detail in thecontext of the medical uses of the invention and its definition andparticularities are herein included by reference.

The person skilled in the art will immediately know which agents areindicated for the treatment of a disease in particular. Almost all theagents that are indicated for the treatment of a pathology characterizedby an increase in expression of TLR-4 and/or an increase in activationof TLR-4 can be comprised in the complex of the invention, although theTLR-4 antagonist agents and anti-inflammatory agents are particularlypreferred. Although numerous types of drugs can be used in the contextof the invention, in a preferred embodiment, the present inventioncontemplates that the drug is selected from the group including, withoutlimitation, TLR-4 antagonists, such as naloxone, naltrexone, LPS,ibudilast, propentofylline, amitriptyline, ketotifen, cyclobenzaprine,mianserin and imipramine; anti-platelet drugs, such as aspirin andclopidogrel; anti-coagulants, such as heparin, acenocumarol, warfarin,dabigatran and rivaroxaban; and antioxidants, such as edaravone.Although it has already been mentioned in the context of detectablereagents, tissue plasminogen activator and its recombinant variants canbe likewise considered as a drug due to their thrombolytic action.

The present invention contemplates that the drug is a nucleic acid.Therefore, in a preferred embodiment the drug is a nucleic acid. Nucleicacids suitable as drugs in the context of the complex of the inventioninclude antisense RNA, antisense DNA and small interfering RNA, whichhave the capability of silencing the expression of genes involved in apathology characterized by an increase in expression of TLR-4 and/or anincrease in activation of TLR-4, including, without limitation, theNFKB1, RIPK3, IFNB1, LY96 (MD-2), IRF3, TLR3, TIRAP (Mal), TICAM1(TRIF), RIPK1, TRAF6, CD14, TRAM, IKBKG (IKK-gamma), IFNA1 and TLR4genes. The term “antisense RNA”, in the context of the presentinvention, refers to a single-stranded RNA the nucleotide sequence ofwhich is complementary for a target messenger RNA, thereby interferingwith the expression of the respective gene. The term “antisense DNA”, inthe context of the present invention, refers to a single-stranded DNAthe nucleotide sequence of which is complementary for a target messengerRNA, thereby interfering with or silencing the expression of therespective gene. The term “small interfering RNA” or “siRNA”, in thecontext of the present invention, refers to a double-stranded RNA with alength of 20 to 25 nucleotides which is highly specific for thenucleotide sequence of its target messenger RNA, thereby interferingwith the expression of the respective gene.

The present invention contemplates that the drug is a peptide.Therefore, in a preferred embodiment the drug is a peptide. The term“peptide”, in the context of the present invention, refers to a shortchain of amino acids bound by peptide bonds. The peptide will compriseat least 2 amino acids, at least 3 amino acids, at least 4 amino acids,at least 5 amino acids, at least 10 amino acids, at least 15 aminoacids, at least amino acids, at least 30 amino acids, at least 40 aminoacids, at least 50 amino acids, at least 60 amino acids, or at least 70amino acids. Suitable for the purposes of this invention are, interalia, peptides with the capability of binding to a target and ofinducing or inhibiting cell signaling. The term “target bindingpeptide”, in the context of the present invention, refers to a peptidecomprising a target binding region. The term “signaling peptide”, in thecontext of the present invention, refers to a peptide with thecapability of provoking cell signaling, such as cell receptor agonistpeptides. The amino acid sequences suitable for target molecule bindinginclude molecular recognition consensus sequences that are well-known inthe art.

In another particular embodiment, the functional group is ananoparticle. The term “nanoparticle”, in the context of the presentinvention, refers to colloidal systems of the spherical type, rod type,polyhedron type, etc., or similar shapes, having a size less than 1micrometer (μm), which are individually found or are found formingorganized structures (dimers, trimers, tetrahedrons, etc.), dispersed ina fluid (aqueous solution). In a particular embodiment, thenanoparticles suitable for putting the invention into practice have asize less than 1 μm, generally comprised between 1 and nanometers (nm),typically between 5 and 500 nm, preferably between about 10 and 150 nm.In a particular embodiment, the nanoparticles of the invention typicallyhave a mean particle diameter ranging from 2 to 50 nm, preferably from 5to 20 nm, more preferably of 13 nm. The mean particle diameter is themaximum mean particle dimension, with the understanding that theparticles are not necessarily spherical. The shape of said nanoparticlescan widely vary; advantageously, said nanoparticles will adopt anyoptically efficient shape such as spheres, rods, stars, cubes,polyhedrons or any other variant as well as complex associations ofseveral particles; in a particular embodiment, the shape of thenanoparticles for putting the invention into practice is spherical orsubstantially spherical. The shape can be suitably evaluated byconventional light or by means of electron microscopy techniques.

Nanoparticles suitable for use in the present invention includepolymeric nanoparticles, lipid nanoparticles and metal nanoparticles.

Polymeric nanoparticles are formed by a polymeric matrix which isattached to the aptamer. Non-limiting examples of biocompatible polymersthat may be useful in the polymeric nanoparticules according to thepresent invention include polyethylenes, polycarbonates, polyanhydrides,polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides,polyacetals, polyethers, polyesters, poly(orthoesters),polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes,polyacrylates, polymethacrylates, polycyanoacrylates, polyureas,polystyrenes, or polyamines, polyglutamate, dextran, polyanhydrides,polyurethanes, polymethacrylates, polyacrylates orpolycyanoacrylates.polydioxanone (PDO), polyhydroxyalkanoate,polyhydroxybutyrate, poly(glycerol sebacate), polyglycolide,polylactide, PLGA, polycaprolactone or combinations thereof.

Alternatively, the nanoparticles of the invention may be lipidnanoparticles such as a liposome or a micelle. Formation of micelles andliposomes from, for example, vesicle-forming lipids, is known in theart. Vesicle-forming lipids refer to lipids that spontaneously formlipid bilayers above their gel-to-liquid crystalline phase transitiontemperature range. Such lipids typically have certain features thatpermit spontaneous bilayer formation, such as close to identicalcross-section areas of their hydrophobic and hydrophilic portionspermitting packing into lamellar phases. Lipids capable of stableincorporation into lipid bilayers, such as cholesterol and its variousanalogs, can be incorporated into the lipid bilayer during bilayerformation. The vesicle-forming lipids are preferably lipids having twohydrocarbon chains, typically acyl chains, and a head group, eitherpolar or nonpolar. There are a variety of synthetic vesicle-forminglipids and naturally-occurring vesicle-forming lipids, including thephospholipids, such as phosphatidylcholine, phosphatidylethanolamine,phosphatidic acid, phosphatidylinositol, and sphingomyelin, where thetwo-hydrocarbon chains are typically between about 14-22 carbon atoms inlength, and either saturated or having varying degrees of unsaturation.The above-described lipids and phospholipids whose acyl chains havevarying degrees of saturation can be obtained commercially or preparedaccording to published methods. Other suitable lipids includephospholipids, sphingolipids, glycolipids, and sterols, such ascholesterol.

The term “liposome” refers to vesicles comprised of one or moreconcentrically ordered lipid bilayers, which encapsulate an aqueousphase. The aqueous phase typically contains the compound to be deliveredto a target site. Upon reaching a target site, the liposome fuses withthe plasma membranes of target cells, i.e. cells expressing TLR-4,thereby releasing the compound into the cytosol. Alternatively, theliposome is endocytosed or otherwise taken in by the target cells as thecontent of a transport vesicle (e.g., an endosome or phagosome). Once inthe transport vesicle, the liposome either degrades or fuses with themembrane of the vesicle and releases its contents. A variety of methodsknown to the skilled person are available for preparing liposomes, suchas sonication, extrusion, high pressure/homogenization, microfluidization, detergent dialysis, calcium-induced fusion of smallliposome vehicles and ether fusion methods, all of which are well knownin the art.

Polymeric and lipidic nanoparticles can additionally include a coatingof a amphiphilic compound that surrounds the polymeric material forminga shell for the particle or a stealth material that can allow theparticles to evade recognition by immune system components and increaseparticle circulation half-life.

Alternatively, the nanoparticles of the invention may be a metalnanoparticle. The term “metal nanoparticle” refers to a nanoparticlecomprising a metal and showing the optical property known as the surfaceplasmon phenomenon, i.e., a plasmonic metal. This phenomenon consists ofthe collective vibration of the electrons of the metal surface,producing an absorption band located in the ultraviolet-visible spectrum(typical of the metal and of the size of the nanoparticles) at thewavelength where the resonance condition occurs in said electrons. Thesurface plasmon of a metal can be determined by means of anyspectroscopic technique known in the state of the art, such as surfaceplasmon resonance (SPR) spectroscopy, whereby the metal atoms aresubjected to an electromagnetic beam or surface plasmon resonancefluorescence spectroscopy (SPFS) based on the detection of the variationof the refractive index of the metal atoms when they are subjected to aphoton beam. As defined herein, a “plasmonic metal” is a metalcharacterized by showing the property of optics known as the surfaceplasmon phenomenon. The variation of the plasmonic response isparticularly evident when several nanoparticles are located close to oneanother, given that this causes the coupling of their respective nearfields, generating a new surface plasmon. In a preferred embodiment,said metal is selected from the group consisting of gold, silver,copper, aluminum, platinum, iron, cobalt, palladium and combinationsthereof.

A preferred embodiment of metal nanoparticles is a core-shellnanoparticle, which contains a metal core and a porous shell. Examplesof core-shell metal nanoparticles include magnetic mesoporous silicananoparticles, which are well-known in the art. Thus, in a particularlypreferred embodiment, the nanoparticle is a magnetic mesoporous silicananoparticle.

The nanoparticles may be functionalized by adding a coating on itssurface. For biological applications, the surface coating should bepolar to give high aqueous solubility and prevent nanoparticleaggregation. In serum or on the cell surface, highly charged coatingspromote non-specific binding, whereas polyethylene glycol linked toterminal hydroxyl or methoxy groups repel non-specific interactions.

Aptamers can be linked to nanoparticles ideally by a covalent link,preferably on the nanoparticle surface. Preferably, aptamers should bepresent in a controlled number per nanoparticle.

Binding between an aptamer of the invention and a functional group forgenerating the complex of the invention can be carried out by means ofconjugation techniques that are well-known by the person skilled in theart. The result is a covalent bond between the aptamer of the inventionand the functional group. The conjugation can involve binding of primaryamines of the 3′ or 5′ ends of the aptamer of the invention to thefunctional group during chemical synthesis of the aptamer.Alternatively, conjugation can be done by means of conventionalcross-linking reactions, having the advantage of the much greaterchemical reactivity of primary alkyl-amine labels with respect to thearyl amines of the nucleotides themselves. Methods of conjugation arewell-known in the art and are based on the use of cross-linkingreagents. The cross-linking reagents contain at least two reactivegroups which target groups such as primary amines, sulfhydryls,aldehydes, carboxyls, hydroxyls, azides and so on and so forth, in themolecule to be conjugated. The cross-linking agents differ in theirchemical specificity, spacer arm length, spacer arm composition,cleavage spacer arm, and structure. For example, conjugation ofcomplexes according to the invention can be carried out directly orthrough a linking moiety, through one or more non-functional groups inthe aptamer and/or the functional group, such as amine, carboxyl,phenyl, thiol or hydroxyl groups. More selective bonds can be achievedby means of the use of a heterobifunctional linker. It is possible touse conventional linkers, such as diisocyanates, diisothiocyanates, bis(hydroxysuccinimide) esters, carbodiimides, maleimide-hydroxysuccinimideesters, glutaraldehyde and the like, or hydrazines and hydrazides, suchas 4-(4-N-maleimidophenyl) butyric acid hydrazide (MPBH).

Another approach consists of labeling the aptamers during synthesis bymeans of PCR using primers labeled, for example, with a fluorophore. Tothat end, there are various commercial establishments available for theperson skilled in the art.

Additionally, in the particular embodiment in which the functional groupis a radionuclide, binding between an aptamer according to the inventionand the radionuclide can be carried out by means of chemicalcoordination, wherein the atoms of the aptamer involved in the bindingdonate electrons to the radionuclide. Coordination reactions arewell-known in the art and will depend on the radionuclide and thereactive group involved in the aptamer.

In Vitro Uses of the Invention A. In Vitro Uses for Detecting TLR-4

The present invention also contemplates in vitro uses of a nucleic acidaptamer with the capability of binding specifically to and inhibitingTLR-4 and comprising a sequence selected from the group consisting ofSEQ ID NO: 1 and SEQ ID NO: 2 or a functionally equivalent variantthereof, and of a complex comprising a nucleic acid aptamer with thecapability of binding specifically to and inhibiting TLR-4 andcomprising a sequence selected from the group consisting of SEQ ID NO: 1and SEQ ID NO: 2 or a functionally equivalent variant thereof and afunctional group, for detecting TLR-4.

Therefore, in another aspect, the present invention refers to an invitro use of a nucleic acid aptamer with the capability of bindingspecifically to and inhibiting TLR-4 and comprising a sequence selectedfrom the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2 or afunctionally equivalent variant thereof for detecting TLR-4.

Therefore, the capability of an aptamer according to the invention ofbinding specifically to TLR-4 can be exploited for the indirectdetection of TLR-4 through the aptamer according to the invention. Forthis purpose, the person skilled in the art will recognize thatsubsequent detection of said aptamer is required. Aptamer detectiontechniques are well-known in the art and include, for example, the useof antibodies or probes specific for the aptamer. Therefore, once theaptamer according to the invention is bound to TLR-4, an antibody orprobe specific for the aptamer, which in turn can be labeled with adetectable reagent, or which can be detected indirectly by means of asecondary antibody or probe, would be applied. The technique used fordetecting TLR-will then depend on the type of detectable reagent, beingable to be techniques based, for example, on fluorimetry, colorimetry orradioactivity.

The term “probe” or “hybridization probe”, in the context of the presentinvention, refers to a variable-length DNA or RNA fragment, generallybetween 10 and bases in length, which is used for detecting the presenceof single-stranded nucleic acids (DNA or RNA) which are complementary tothe sequence in the probe. The probe is hybridized to the targetsingle-stranded nucleic acid, the base sequence of which allows basepairing due to complementarity between the probe and the target nucleicacid. For detecting hybridization of the probe to its target sequence,the probe is labeled with a detectable reagent, such as a radionuclide,a fluorophore or digoxigenin, inter alia.

The detection of TLR-4 with the aptamer of the invention can be carriedout by means of in vitro binding assays, such as the enzyme-linkedoligonucleotide assay (ELONA), the enzyme-linked aptamer sorbent assay(ELASA), precipitation and quantitative PCR (qPCR), gel mobility shiftassay, Western Blotting, surface plasmon resonance (SPR), kineticcapillary electrophoresis, the fluorescence binding assay,aptahistochemistry, aptacytochemistry, fluorescence microscopy or flowcytometry.

In another particular embodiment of the in vitro uses of the invention,the detection of TLR-4 is performed by means of a method selected fromthe group consisting of ELONA, aptacytochemistry, aptahistochemistry andflow cytometry.

The term “ELONA” or “enzyme-linked oligonucleotide assay”, in thecontext of the present invention, refers to a technique analogous toenzyme-linked immunosorbent assay (ELISA), wherein the antibody that isused for detecting the molecule of interest, in this case TLR-4, isexchanged for a detection aptamer specific for said molecule. The ELISAassay is based on the use of antigens or antibody labeled, for example,with enzymes, such that the complexes formed between the target antigenand the labeled antibody are enzymatically active complexes. Since oneof the components, in this case the antigen, is immobilized in acarrier, the antigen:antibody complexes are immobilized to the carrierand can therefore be detected by means of the addition of a substratespecific for the enzyme. In the case of ELONA, the detection aptamer canbe covalently bound to an enzyme, or it can be detected by itself by asecondary antibody specific for the aptamer that is conjugated to anenzyme. Said enzyme catalyzes the transformation of a specific substrateto produce a visible signal. This technique can be modified to exchangethe enzyme for another detectable reagent, such as a fluorophore. Theterms ELONA and ELASA, or enzyme-linked aptamer sorbent assay, are usedinterchangeably herein. In a preferred embodiment the detection of TLR-4is performed by means of ELONA.

In an analogous manner, the terms “aptacytochemistry” and“aptahistochemistry”, in the context of the present invention, refer totechniques analogous to immunocytochemistry and immunohistochemistry forthe detection of TLR-4 on cells and histological sections, respectively,wherein the antibody that is used for detecting the molecule ofinterest, in this case TLR-4, is exchanged for an aptamer specific forsaid molecule. The detection aptamer can be bound covalently to anenzyme, or it can be detected by itself by a secondary antibody specificfor the aptamer that is conjugated to an enzyme. Said enzyme catalyzesthe transformation of a specific substrate to produce a visible signal.This technique can be modified to exchange the enzyme for anotherdetectable reagent, such as a fluorophore. In a preferred embodiment,the detection of TLR-4 is performed by means of aptacytochemistry. Inanother preferred embodiment, the detection of TLR-4 is performed bymeans of aptahistochemistry.

Alternatively, the person skilled in the art will recognize that thesetechniques (ELONA, aptacytochemistry, aptahistochemistry) can be adaptedfor exchanging the detection antibody for a probe specific for theaptamer.

The term “flow cytometry”, in the context of the present invention,refers to a cell analysis technique that involves measuring thefluorescence and light dispersion features that the cells have as theypass through a ray of light. In addition to light dispersion, if priorto analysis the cells are placed in the presence of aptamers labeledwith fluorescent molecules, it is possible to evaluate which cells haveantigens complementary to the aptamers used. The detection offluorescence is performed with flow cytofluorimeters (known as“cytometers” or “FACS” (fluorescence-activated cell sorter)). Thistechnique, like the preceding techniques, was initially developed foruse with fluorescently labeled antibodies but can be readily adapted foruse with the aptamer of the invention.

As the person skilled in the art will understand, a complex comprising anucleic acid aptamer with the capability of binding specifically to andinhibiting TLR-4 and comprising a sequence selected from the groupconsisting of SEQ ID NO: 1 and SEQ ID NO: 2 or a functionally equivalentvariant thereof and a detectable reagent is particularly advantageousfor detecting TLR-4, since said detectable reagent enables the detectionof the aptamer comprised in the complex when it is bound to TLR-4. Thetechnique used for detecting TLR-4 will depend on the type of detectablereagent, being able to be techniques based, for example, on fluorimetry,colorimetry or radioactivity.

Therefore, in another aspect, the present invention relates to a complexcomprising a nucleic acid aptamer with the capability of bindingspecifically to and inhibiting TLR-4 and comprising a sequence selectedfrom the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2 or afunctionally equivalent variant thereof and a functional group fordetecting TLR-4.

In a particular embodiment, the functional group is a detectablereagent.

In another particular embodiment of the in vitro uses of the invention,the detection of TLR-4 is performed by means of a method selected fromthe group consisting of ELONA, aptacytochemistry, aptahistochemistry andflow cytometry.

The terms “aptamer”, “TLR-4”, “functionally equivalent variant”,“complex”, “functional group”, “detectable reagent”, “ELONA”,“aptacytochemistry”, “aptahistochemistry” and “flow cytometry” have beendescribed in detail above and their definitions and particularities areherein included by reference.

Given that the ELISA, immunocytochemical, immunohistochemical and flowcytometry techniques are well-known in the art, the person skilled inthe art could make the adaptations required for exchanging the antibodyfor the aptamer or complex according to the invention without having toconduct undue experimentation.

B. In Vitro Uses for Inhibiting TLR-4

As described above, a nucleic acid aptamer with the capability ofbinding specifically to and inhibiting TLR-4 and comprising a sequenceselected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2 or afunctionally equivalent variant thereof can inhibit the activity ofTLR-4, reducing the levels of pro-inflammatory cytokines released as aresult of the activation thereof. The present invention alsocontemplates in vitro uses of a nucleic acid aptamer with the capabilityof binding specifically to and inhibiting TLR-4 and comprising asequence selected from the group consisting of SEQ ID NO: 1 and SEQ IDNO: 2 or a functionally equivalent variant thereof, and of a complexcomprising a nucleic acid aptamer with the capability of bindingspecifically to and inhibiting TLR-4 and comprising a sequence selectedfrom the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2 or afunctionally equivalent variant thereof and a functional group, forinhibiting TLR-4.

Therefore, in another aspect, the present invention relates to an invitro use of a nucleic acid aptamer with the capability of bindingspecifically to and inhibiting TLR-4 and comprising a sequence selectedfrom the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2 or afunctionally equivalent variant thereof for inhibiting TLR-4.

In another aspect, the present invention relates to an in vitro use of acomplex comprising a nucleic acid aptamer with the capability of bindingspecifically to and inhibiting TLR-4 and comprising a sequence selectedfrom the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2 or afunctionally equivalent variant thereof and a functional group, forinhibiting TLR-4.

In Vitro Methods of the Invention A. In Vitro Methods for the Detectionof TLR-4

In another aspect, the present invention relates to an in vitro methodfor the detection of TLR-4 in a sample, hereinafter “the first in vitromethod for the detection of TLR-4 of the invention” comprising

-   -   i) contacting said sample with a nucleic acid aptamer with the        capability of binding specifically to and inhibiting TLR-4 and        comprising a sequence selected from the group consisting of SEQ        ID NO: 1 and SEQ ID NO: 2 or a functionally equivalent variant        thereof,    -   ii) separating the aptamer not bound to TLR-4, and    -   iii) detecting the presence of the aptamer bound to the TLR-4        present in the sample.

In a first step, the first in vitro method for detection of theinvention comprises contacting said sample with a nucleic acid aptamerwith the capability of binding specifically to and inhibiting TLR-4 andcomprising a sequence selected from the group consisting of SEQ ID NO: 1and SEQ ID NO: 2 or a functionally equivalent variant thereof.

The terms “aptamer”, “TLR-4” and “functionally equivalent variant” havebeen described in detail above and their definitions and particularitiesare herein included by reference.

The term “sample” or “biological sample”, in the context of the presentinvention, refers to a cell culture or to isolated biological materialfrom a subject. The biological sample can contain any biologicalmaterial suitable for detecting the desired biomarker and can comprisecells and/or non-cellular material from the subject. The sample can beisolated from any suitable tissue or biological fluid such as, forexample, blood, plasma, serum, urine, cerebrospinal fluid (CSF), heart,brain. The samples used for the detection of TLR-4 are preferablybiological fluids.

Alternatively, the samples are biofluid samples. The terms “biologicalfluid” and “biofluid” are used interchangeably herein and refer toaqueous fluids of a biological origin. The biofluid can be obtained fromanywhere (such as blood, plasma, serum, urine, bile, cerebrospinalfluid, vitreous or aqueous humor, or any bodily secretion), an exudate(such as the fluid obtained from an abscess or any other site ofinfection or inflammation), or the fluid obtained from a joint (such asa normal joint or a joint affected by a disease such as rheumatoidarthritis). The biofluids used for the detection of TLR-4 are preferablyblood, plasma, serum or cerebrospinal fluid samples.

The aptamer according to the invention is applied on the sample in abuffer suitable for allowing the binding of the aptamer to the TLR-4molecules that may be present in the sample. Non-limiting examples ofbuffers suitable for allowing the binding of the aptamer of theinvention and TLR-4 include PBS, TBS, phosphate buffer and citratebuffer. Preferably, these buffers contain 1 mM MgCl₂. The amount ofaptamer required for detecting the TLR-4 molecules present in the samplewill depend on both the size of the sample and on the amount of TLR-4present therein, and it could be readily determined by optimizationmethods commonly used in the art. By way of indication, the aptamerconcentration is at least 1 fM, at least 10 fM, at least 100 fM, atleast 1 pM, at least 10 pM, at least 100 pM, at least 1 nM, at least 10nM, at least 100 nM, at least 1 μM, at least 10 μM, at least 100 μM ormore. Preferably, the aptamer concentration is between 100 fM and 1 μM,more preferably between 1 pM and 100 nM, even more preferably between100 pM and 1 nM.

The aptamer is incubated with the sample at a suitable temperature andfor a time sufficient for allowing the binding of the aptamer to theTLR-4 molecules that may be present in the sample. The temperature ispreferably between 20° C. and 37° C. By way of indication, the aptamerwill be incubated with the sample for at least 5 min, at least 10 min,at least 15 min, at least, 20 min, at least 30 min, at least 60 min, atleast 120 min or more.

Once the aptamer has bound to the TLR-4 molecules that may be present inthe sample, in a second step the sample is washed to remove the aptamermolecules that have not bound to TLR-4.

In a third step, the presence of the aptamer bound to the TLR-4 presentin the sample is detected. Since the aptamer of the invention is not byitself a detectable molecule, the step of detection is a step ofindirect detection through a second detectable molecule which bindsspecifically to the aptamer. The detection of the aptamer bound to TLR-4can be carried out with virtually any known antibody or reagent thatbinds with high affinity to the aptamer of the invention. Nevertheless,the use of an antibody specific for the aptamer, for example, polyclonalserum, hybridoma supernatant, monoclonal or humanized antibodies andfragments thereof, is preferred. Said antibody specific for the aptameris suitably labeled with a detectable reagent. The term “detectablereagent” has been described in detail above and its definition andparticularities are herein included by reference. Said reagent can bedetected by means of fluorimetry or colorimetry using apparatusessuitable for the type of reagents and the type of sample, which areknown by the person skilled in the art. By way of example, the samplewith the aptamer bound to the TLR-4 molecules present is incubated withan antibody specific for the aptamer that is conjugated with an enzyme,in conditions similar to the conditions of incubation with the aptamer,and the TLR-4-aptamer-antibody complexes are detected with the additionof a substrate that is converted by the enzyme into a detectableproduct, for example, by means of fluorimetry in a fluorescencemicroscope or by colorimetry in a spectrophotometer. Alternatively,detection can be done in an analogous manner by means of the use of aprobe specific for the aptamer suitably labeled with a detectablereagent.

The person skilled in the art will recognize that the first in vitromethod of the invention can be carried out as part of detectiontechniques such as ELONA, ELASA, precipitation and qPCR, gel mobilityshift assay, Western Blotting, surface plasmon resonance, kineticcapillary electrophoresis, fluorescence binding assay,aptahistochemistry, aptacytochemistry, fluorescence microscopy or flowcytometry.

Alternatively, the aptamer according to the invention can be bound to afunctional group that is part of a complex according to the invention.Therefore, in another aspect, the present invention relates to an invitro method for the detection of TLR-4 in a sample, hereinafter “thesecond in vitro method for the detection of TLR-4 of the invention”,comprising

-   -   i) contacting said sample with a complex comprising a nucleic        acid aptamer with the capability of binding specifically to and        inhibiting TLR-4 and comprising a sequence selected from the        group consisting of SEQ ID NO: 1 and SEQ ID NO: 2 or a        functionally equivalent variant thereof and a functional group,    -   ii) separating the aptamer or complex not bound to TLR-4, and    -   iii) detecting the presence of the complex bound to the TLR-4        present in the sample.

The terms “aptamer”, “TLR-4”, “functionally equivalent variant”, and“sample” have been described in detail above and their definitions andparticularities are herein included by reference. Likewise, theparticularities of the first and second steps of the first in vitromethod for detection of the invention also apply to the second in vitromethod for detection of the invention and are likewise included byreference.

The third step of the second in vitro method for detection of theinvention comprises detecting the presence of the complex of theinvention bound to the TLR-4 present in the sample. The detection of thecomplex according to the invention can be carried out with virtually anyknown antibody or reagent that binds with high affinity to the aptamerof the invention or to the functional group. The detection of theaptamer of the invention has been described in detail in the context ofthe first in vitro method for detection of the invention. Likewise, inrelation to the functional group, the detection can also be carried outwith virtually any known antibody or reagent that binds with highaffinity to said functional group. For this reason, it is particularlyappropriate for the second in vitro method for detection of theinvention that the functional group is a detectable reagent.

In a particular embodiment the functional group is a detectable reagentselected from the group formed by radionuclides, fluorophores, proteinsand haptenes.

The terms “radionuclide”, “fluorophore”, “detectable protein” and“haptene” have been described in detail above and their definitions andparticularities are herein included by reference.

As the person skilled in the art will understand, the detectablereagents contemplated by the present invention can be divided betweenthe reagents which are directly detectable by themselves, such asradionuclides or fluorophores, and the reagents which are indirectlydetectable, such as proteins or haptenes.

In a preferred embodiment, the detectable reagent is a radionuclide andthe detection is performed by detection of the radiation emitted by theradionuclide. Said radiation will depend on the type of radionuclide,being able to be an a particle emission, β particle emission or y typeemission. For this purpose, detection techniques suitable for differentradionuclides are well-known. By way of example, the emission emitted by¹²³I can be detected by a gamma camera.

In another preferred embodiment, the detectable reagent is a fluorophoreand the detection is performed by detection of the fluorescence emittedby the fluorophore. The use of a fluorophore requires the priorexcitation thereof with a wavelength within its excitation spectrum,which causes emission at a different wavelength. The excitation andemission wavelengths of the fluorophores contemplated in the presentinvention are part of the state of the art. The fluorescence emitted canbe detected, for example, through fluorimetry techniques by using afluorescence spectrophotometer or a fluorescence microscope.

In another preferred embodiment, the detectable reagent is a protein.Said protein could be detected depending on the type of protein used.For example:

-   -   an enzyme requires the addition of its specific substrate which        will be detectable by colorimetry, chemiluminescence or        fluorimetry;    -   a fluorescent protein, like a fluorophore, requires excitation        at a wavelength suitable for being detectable by fluorimetry        (for example, the wavelengths included in Table 1);    -   an antigen or an haptene requires an antibody or another        molecule that specifically recognizes it. In order to be        detected, said antibody or molecule specific for the        antigen/haptene must be labeled, for example, with an enzyme,        and the detection will depend on the type of labeling.

The person skilled in the art will recognize that the second in vitromethod of the invention can be carried out as part of detectiontechniques such as ELONA, ELASA, precipitation and qPCR, gel mobilityshift assay, Western Blotting, surface plasmon resonance, kineticcapillary electrophoresis, fluorescence binding assay,aptahistochemistry, aptacytochemistry, fluorescence microscopy or flowcytometry.

In a particular embodiment, the detection of TLR-4 is performed by meansof fluorescence.

B. In Vitro Methods for the Inhibition of TLR-4

In another aspect, the present invention relates to an in vitro methodfor inhibiting TLR-4 in a sample, hereinafter “the first in vitro methodfor the inhibition of TLR-4 of the invention”, which comprisescontacting a sample comprising TLR-4 with a nucleic acid aptamer withthe capability of binding specifically to and inhibiting TLR-4 andcomprising a sequence selected from the group consisting of SEQ ID NO: 1and SEQ ID NO: 2 or a functionally equivalent variant thereof, inconditions suitable for inhibiting TLR-4.

The terms “aptamer”, “TLR-4”, “functionally equivalent variant”,“inhibition of TLR-4” and “sample” have been described in detail aboveand their definitions and particularities are herein included byreference.

In a particular embodiment, the TLR-4 of the sample is comprised inliving cells.

The first in vitro method for the inhibition of TLR-4 of the inventioncomprises contacting a sample comprising TLR-4 with an aptamer accordingto the invention in conditions suitable for inhibiting TLR-4. To thatend, the aptamer of the invention is applied on the sample and it mustbind to TLR-4.

The term “conditions suitable for inhibiting TLR-4”, in the context ofthe present invention, refers to the incubation conditions that allowthe binding of the aptamer of invention to TLR-4 and the subsequentinhibition thereof. These conditions include the composition of thebuffer in which the aptamer of the invention is applied on the sample,the amount of aptamer, the incubation time and the incubationtemperature. Non-limiting examples of buffers suitable for allowing thebinding of the aptamer of the invention to TLR-4 and the inhibitionthereof include PBS, TBS, phosphate buffer and citrate buffer.Preferably, these buffers contain 1 mM MgCl₂. The amount of aptamerrequired for detecting the TLR-4 molecules present in the sample willdepend both on the size of the sample and on the amount of TLR-4 presenttherein, and it could be readily determined by optimization methodscommonly used in the art. By way of indication, the aptamerconcentration is at least 1 fM, at least 10 fM, at least 100 fM, atleast 1 pM, at least 10 pM, at least 100 pM, at least 1 nM, at least 10nM, at least 100 nM, at least 1 μM, at least 10 μM, at least 100 μM ormore. Preferably, the aptamer concentration is between 100 fM and 1 μM,more preferably between 1 pM and 100 nM, even more preferably between100 pM and 1 nM.

The aptamer is incubated with the sample at a suitable temperature andfor a time sufficient for allowing the binding of the aptamer to theTLR-4 molecules that may be present in the sample. The temperature ispreferably between 20° C. and 37° C., more preferably 37° C. By way ofindication, the aptamer will be incubated with the sample for at least 5min, at least 10 min, at least 15 min, at least 20 min, at least 30 min,at least 60 min, at least 120 min or more.

In another aspect, the present invention relates to an in vitro methodfor inhibiting TLR-4 in a sample, hereinafter “the second in vitromethod for the inhibition of TLR-4 of the invention”, which comprisescontacting a sample comprising TLR-4 with a complex comprising a nucleicacid aptamer with the capability of binding specifically to andinhibiting TLR-4 and comprising a sequence selected from the groupconsisting of SEQ ID NO: 1 and SEQ ID NO: 2 or a functionally equivalentvariant thereof and a functional group in conditions suitable forinhibiting TLR-4.

The terms “aptamer”, “TLR-4”, “functionally equivalent variant”,“inhibition of TLR-4”, “sample” and “conditions suitable for inhibitingTLR-4” have been described in detail above and their definitions andparticularities are herein included by reference.

Medical Uses of the Invention

The authors of the present invention have demonstrated that the aptamerof the invention is able to block or inhibit the volume of infarctionproduced in a model of stroke induced in animals used in experiments, asdescribed in Example 4. Therefore, the capability of the aptamer of theinvention of binding specifically to and inhibiting TLR-4 renders ituseful from a therapeutic viewpoint. It is apparent that the therapeuticeffect obtained with the aptamer of the invention can be complementedwith a functional group with therapeutic activity, as described in thecontext of the complex of the invention. Accordingly, the presentinvention contemplates the medical uses of the aptamer of the inventionand of the complex of the invention.

A. Medical Uses of the Aptamer of the Invention

In another aspect, the present invention relates to a nucleic acidaptamer with the capability of binding specifically to and inhibitingTLR-4 and comprising a sequence selected from the group consisting ofSEQ ID NO: 1 and SEQ ID NO: 2 or a functionally equivalent variantthereof for use in medicine.

In another aspect, the present invention relates to a nucleic acidaptamer with the capability of binding specifically to and inhibitingTLR-4 and comprising a sequence selected from the group consisting ofSEQ ID NO: 1 and SEQ ID NO: 2 or a functionally equivalent variantthereof for use in manufacturing a drug for the treatment of a pathologycharacterized by an increase in expression of TLR-4 and/or an increasein activation of TLR-4.

Alternatively, it can be expressed as the use of a nucleic acid aptamerwith the capability of binding specifically to and inhibiting TLR-4 andcomprising a sequence selected from the group consisting of SEQ ID NO: 1and SEQ ID NO: 2 or a functionally equivalent variant thereof for use inthe treatment of a pathology characterized by an increase in expressionof TLR-4 and/or an increase in activation of TLR-4.

Alternatively, it can be expressed as an in vivo method for thetreatment of a pathology characterized by an increase in expression ofTLR-4 and/or an increase in activation of TLR-4 in a subject, comprisingthe administration to said subject of a therapeutically effective amountof a nucleic acid aptamer with the capability of binding specifically toand inhibiting TLR-4 and comprising a sequence selected from the groupconsisting of SEQ ID NO: 1 and SEQ ID NO: 2 or a functionally equivalentvariant thereof to said subject.

According to the invention, a nucleic acid aptamer with the capabilityof binding specifically to and inhibiting TLR-4 and comprising asequence selected from the group consisting of SEQ ID NO: 1 and SEQ IDNO: 2 or a functionally equivalent variant thereof binds specifically toTLR-4 on the surface of a target cell. When the aptamer of the inventionis contacted with TLR-4, it inhibits its activity, resulting in areduction or interruption in the release of pro-inflammatory cytokinessuch as IL-1, IL-8, TNF-alpha and IL-12.

The term “aptamer” has been described in detail in relation to theDefinitions and the Aptamer specific for TLR-4 (supra) and itsdefinitions and particularities likewise apply in the context of thecomplex of the invention.

The term “treatment” or “therapy”, in the context of the presentinvention, refers to the clinical intervention in an attempt to prevent,cure, delay, reduce the seriousness of, or improve one or more symptomsof the pathology characterized by an increase in expression of TLR-4and/or an increase in activation of TLR-4, or for the purpose ofprolonging survival of a patient beyond what is expected in the absenceof such treatment.

The term “target cell”, in the context of the present invention, refersto the particular cell that expresses TLR-4, including, inter alia,myeloid lineage cells such as monocytes, macrophages, microglia cells,granulocytes and immature dendritic cells, as well as cells of otherlineages such as neurons, etc. In a particular embodiment, the targetcell is a monocyte or a macrophage. In another particular embodiment,the target cell is a microglia cell. In another particular embodiment,the target cell is a granulocyte. In another particular embodiment, thetarget cell is an immature dendritic cell. In another particularembodiment, the target cell is a neuron.

In a particular embodiment, the target cell is a mammal cell. In anotherpreferred embodiment, the mammal cell is a human cell.

The term “subject” or “individual” refers to a member of a mammalspecies, and includes, but is not limited to, domestic animals andprimates, including humans; the subject is preferably a male or femalehuman of any age or race.

The term “a therapeutically effective amount”, in the context of thepresent invention, refers to the amount of the aptamer of the inventionrequired for achieving a prevention, cure, delay, reduction of theseriousness of, or improvement of one or more observable symptoms of thepathology characterized by an increase in expression of TLR-4 and/or anincrease in activation of TLR-4.

The term “pathology characterized by an increase in expression of TLR-4and/or an increase in activation of TLR-4”, in the context of thepresent invention, refers to a pathology in which the cells that expressTLR-4 show an increase in expression of TLR-4 and/or an increase inactivation of TLR-4, and/or a pathology in which there is an increase inthe amount of cells that express TLR-4, with respect to normal orreference physiological conditions or reference values, and in whichsaid cells are directly or indirectly involved regardless of whether ornot TLR-4 is responsible for the disease. Given that activation of TLR-4produces a signaling cascade resulting in the release of inflammatorycytokines such as IL-1, IL-8, TNF-alpha and IL-12, causing inflammationand cell damage, the pathology characterized by an increase inexpression of TLR-4 and/or an increase in activation of TLR-4 canfurthermore be characterized by having an inflammatory component.

In a particular embodiment, said pathology characterized by an increasein expression of TLR-4 and/or an increase in activation of TLR-4 isselected from the group consisting of, inter alia, stroke, acutemyocardial infarction, sepsis, atherosclerosis, multiple sclerosis,rheumatoid arthritis, a retinal degenerative disease, and drugaddiction.

In a preferred embodiment, said pathology characterized by an increasein expression of TLR-4 and/or an increase in activation of TLR-4 is astroke. The term “stroke” or “cerebrovascular disease” or “cerebralinfarction” or “apoplexy”, in the context of the present invention,refers to a pathology characterized by a neurological deficit caused byan important decrease in cerebral blood flow, in an abnormally abruptmanner (ischemic stroke), or due to hemorrhaging caused by the ruptureof a vessel of the brain (hemorrhagic stroke). In ischemic stroke, bloodirrigation is lost due to the sudden and immediate interruption of bloodflow due to occlusion of any of the arteries irrigating the brain mass,which generates the appearance of an infarcted area. Artery occlusion isgenerally due to atherosclerosis or an embolus (cerebral embolism) thatcomes from another location, fundamentally the heart or other arteries.In hemorrhagic stroke, the rupture of a blood vessel in the brainoccurs, depriving the area of the brain that depends on that artery ofblood. In addition the blood that flows out compresses brain structures,including other blood vessels, which increases the affected area byischemia secondary to the intracerebral hemorrhage.

In a preferred embodiment, said pathology characterized by an increasein expression of TLR-4 and/or an increase in activation of TLR-4 isacute myocardial infarction. The term “acute myocardial infarction” or“infarction” or “heart attack”, in the context of the present invention,refers to a pathology characterized by insufficient blood supply, withtissue damage, in an area of the heart, caused by an obstruction in oneof the coronary arteries. Ischemia or deficient oxygen supply resultingfrom such obstruction causes angina pectoris, which if recannulated soonenough, does not cause death of heart tissue, whereas if this anoxia ismaintained, the myocardium becomes injured and necrosis, i.e.,infarction, ultimately occurs.

In a preferred embodiment, said pathology characterized by an increasein expression of TLR-4 and/or an increase in activation of TLR-4 issepsis. The term “sepsis” or “septicemia”, in the context of the presentinvention, refers to the systemic inflammatory response syndrome (SIRS)caused by a generally serious infection. This reaction of the organismoccurs in response to the presence of pathogenic microorganisms in anytissue or fluid of the organism, and is caused by the action of theimmune system itself, which releases pro-inflammatory substances whichstart up the SIRS. It is characterized by the presence of at least twoof the following criteria: fever, hyperthermia, tachypnea, tachycardiaand leukocytosis.

In a preferred embodiment, said pathology characterized by an increasein expression of TLR-4 and/or an increase in activation of TLR-4 isatherosclerosis. The term “atherosclerosis”, in the context of thepresent invention, refers to a syndrome or pathology characterized bythe deposition and infiltration of lipid substances in the walls ofmedium- and thick-sized arteries. The cells of the arterial wallinterpret this deposition as an invasion and activate circulatingmonocytes of the immune system, which penetrate the arterial wall, areconverted into macrophages and start to phagocyte LDL particles,generating an inflammatory process. Inflammation in turn causes themultiplication and migration of the smooth muscle cells of the wall,which gradually cause narrowing of the arterial diameter. The specificthickening is referred to as an atheromatous plaque. It is the mostcommon form of arteriosclerosis. The diseases forming atherosclerosissyndrome and characterized by involvement of the arteries throughatheromatous plaques, and accordingly obstruction of blood flow orischemia, depending on the artery of the organ involved, are:

-   -   Ischemic heart disease, the maximum representative thereof being        acute myocardial infarction, in the heart.    -   Cerebrovascular disease, in the form of stroke or cerebral        thrombosis or cerebral hemorrhage, in the central nervous        system.    -   Intermittent claudication, the maximum seriousness thereof being        acute arterial ischemia of the lower limbs.    -   Erectile dysfunction: this is the principal cause of impotency        in people over 40 years of age.    -   Ischemic colitis, which is an area of inflammation (irritation        and swelling) caused by interference with the blood flow to the        colon (large intestine), in the arteries of the intestines.    -   Aortic aneurism, the maximum seriousness thereof being aortic        dissection.

In a preferred embodiment, said pathology characterized by an increasein expression of TLR-4 and/or an increase in activation of TLR-4 ismultiple sclerosis. The term “multiple sclerosis”, in the context of thepresent invention, refers to a pathology characterized by the onset ofdemyelinating, neurodegenerative and chronic lesions of the centralnervous system. Its causes are currently unknown, although theinvolvement of various autoimmune mechanisms has been demonstrated. Inmultiple sclerosis patients, lymphocytes cross the blood-brain barrierto affect the myelin, while an inflammatory process aided by macrophagesand neuroglia cells occurs.

In a preferred embodiment, said pathology characterized by an increasein expression of TLR-4 and/or an increase in activation of TLR-4 isrheumatoid arthritis. The term “rheumatoid arthritis”, in the context ofthe present invention, refers to a systemic autoimmune inflammatorypathology, characterized by causing persistent synovitis of the joints,causing their progressive destruction, generating different degrees ofdeformity and functional disability. The process starts with theintervention of humoral and cell factors, particularly CD4 T-cells,which generate inflammation mediating molecules, attract and activateperipheral blood cells, causing proliferation and activation of thesynoviocytes, invading and destroying joint cartilage, subchondral bone,tendons and ligaments.

In a preferred embodiment, said pathology characterized by an increasein expression of TLR-4 and/or an increase in activation of TLR-4 is aretinal degenerative disease. The term “retinal degenerative disease”,in the context of the present invention, refers to a disease or disordercharacterized by a degeneration of the retina, which may be the resultof retinal inflammation. TLR-4-mediated microglial activation has beenshown to make a contribution to the process of retinal inflammation.Major retinal degenerative diseases include:

-   -   Age-related macular degeneration (AMD), which results in a loss        of vision in the center of the visual field (the macula) because        of damage to the retina. It occurs in “dry” and “wet” forms: in        the dry (nonexudative) form, cellular debris called drusen        accumulates between the retina and the choroid, causing atrophy        and scarring to the retina; in the wet (exudative) form, which        is more severe, blood vessels grow up from the choroid behind        the retina which can leak exudate and fluid and also cause        hemorrhaging.    -   Stargardt disease, or fundus flavimaculatus, is an inherited        form of juvenile macular degeneration that causes progressive        vision loss usually to the point of legal blindness. The onset        of symptoms usually appears between the ages of 6 and 13 years        old (average of about 16-18 years). Symptoms typically develop        by 20 years of age, and include wavy vision, blind spots,        blurriness, impaired color vision, and difficulty adapting to        dim lighting.    -   Retinitis pigmentosa (RP), which is an inherited, degenerative        eye disease that causes severe vision impairment due to the        progressive degeneration of the rod photoreceptor cells in the        retina. Patients in the early stages of RP first notice        compromised peripheral and dim light vision due to the decline        of the rod photoreceptors. The progressive rod degeneration is        later followed by abnormalities in the adjacent retinal pigment        epithelium and the deterioration of cone photoreceptor cells. As        peripheral vision becomes increasingly compromised, patients        experience progressive “tunnel vision” and eventual blindness.    -   Other genetic diseases such as choroideremia, Leber congenital        amaurosis, retinoschisis juvenile, Usher disease, and Bardet        Biedl.

In a particularly preferred embodiment, the retinal degenerative diseaseis selected from the group consisting of AMD, Stargardt disease, RP,choroideremia, Leber congenital amaurosis, retinoschisis juvenile, Usherdisease, and Bardet Biedl. In a more preferred embodiment, the retinaldegenerative disease is AMD. In another more preferred embodiment, theretinal degenerative disease is Stargardt disease. In another morepreferred embodiment, the retinal degenerative disease is RP. In anothermore preferred embodiment, the retinal degenerative disease ischoroideremia. In another more preferred embodiment, the retinaldegenerative disease is Leber congenital amaurosis. In another morepreferred embodiment, the retinal degenerative disease is Usher disease.In another more preferred embodiment, the retinal degenerative diseaseis Bardet Biedl.

In a preferred embodiment, said pathology characterized by an increasein expression of TLR-4 and/or an increase in activation of TLR-4 is adrug addiction. The term “drug addiction” or “drug dependence”, in thecontext of the present invention, refers to a disorder or pathologycaused by the frequent use of addictive substances called drugs.According to ICD-10 (World Health Organization, 2005), in order to bediagnosed as such, the drug dependence must present three or more of thefollowing criteria, which refer both to aspects related to physicaldependence and to psychological dependence, in a 12-month period:

-   -   strong craving to consume the substance,    -   difficulties in controlling said consumption,    -   withdrawal syndrome when consumption is discontinued or reduced,    -   tolerance,    -   progressive abandonment of interests other than substance        consumption,    -   increase in time invested in activities related to obtaining the        substance or to recovering from its effects,    -   persistence in the use of the substance despite clearly        perceiving its harmful effects.

For the administration to a subject of a nucleic acid aptamer with thecapability of binding specifically to and inhibiting TLR-4 andcomprising a sequence selected from the group consisting of SEQ ID NO: 1and SEQ ID NO: 2 or a functionally equivalent variant thereof, saidaptamer will be formulated in a suitable pharmaceutical composition. Thedetails of said pharmaceutical composition are discussed below.

B. Medical Uses of the Complex of the Invention

The complexes according to the present invention comprising a nucleicacid aptamer with the capability of binding specifically to andinhibiting TLR-4 and comprising a sequence selected from the groupconsisting of SEQ ID NO: 1 and SEQ ID NO: 2 or a functionally equivalentvariant thereof and a functional group according to the invention cancomprise, as a functional group, a drug suitable for the treatment of apathology characterized by an increase in expression of TLR-4 and/or anincrease in activation of TLR-4. The dual objective of (i) inhibitingthe activity of TLR-4 and (ii) directing the drug in a specific mannerto its site of action, is therefore achieved.

Therefore, in another aspect, the present invention refers to a complexcomprising a nucleic acid aptamer with the capability of bindingspecifically to and inhibiting TLR-4 and comprising a sequence selectedfrom the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2 or afunctionally equivalent variant thereof and a functional group for usein medicine.

In another aspect, the present invention relates to a complex comprisinga nucleic acid aptamer with the capability of binding specifically toand inhibiting TLR-4 and comprising a sequence selected from the groupconsisting of SEQ ID NO: 1 and SEQ ID NO: 2 or a functionally equivalentvariant thereof and a functional group for use in manufacturing a drugfor the treatment of a pathology characterized by an increase inexpression of TLR-4 and/or an increase in activation of TLR-4.

Alternatively, it can be expressed as the use of a complex comprising anucleic acid aptamer with the capability of binding specifically to andinhibiting TLR-4 and comprising a sequence selected from the groupconsisting of SEQ ID NO: 1 and SEQ ID NO: 2 or a functionally equivalentvariant thereof and a functional group for use in the treatment of apathology characterized by an increase in expression of TLR-4 and/or anincrease in activation of TLR-4.

Alternatively, it can be expressed as method of treatment of a pathologycharacterized by an increase in expression of TLR-4 and/or an increasein activation of TLR-4 in a subject, comprising the administration tosaid subject of a therapeutically effective amount of a complexcomprising a nucleic acid aptamer with the capability of bindingspecifically to and inhibiting TLR-4 and comprising a sequence selectedfrom the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2 or afunctionally equivalent variant thereof and a functional group to saidsubject.

According to the invention, a complex comprising a nucleic acid aptamerwith the capability of binding specifically to and inhibiting TLR-4 andcomprising a sequence selected from the group consisting of SEQ ID NO: 1and SEQ ID NO: 2 or a functionally equivalent variant thereof and a drugbinds specifically to TLR-4 in the surface of a target cell. When theaptamer of the invention is contacted with TLR-4, it inhibits itsactivity, resulting in a reduction or interruption in the release ofpro-inflammatory cytokines such as IL-1, IL-8, TNF-alpha and IL-12, andthe drug exerts its function on the cell or on the environment wheresaid cell is located.

The terms “aptamer”, “TLR-4”, “functionally equivalent variant”,“complex”, “functional group”, “drug”, “treatment”, “therapeuticallyeffective amount”, “subject”, “target cell” and “pathology characterizedby an increase in expression of TLR-4 and/or an increase in activationof TLR-4” have been described in detail above and their definitions andparticularities are herein included by reference.

Suitable drugs that can be used as functional groups in the complexesformed with a nucleic acid aptamer with the capability of bindingspecifically to and inhibiting TLR-4 include, without limitation,antagonists of TLR-4, such as naloxone, naltrexone, LPS, ibudilast,propentofylline, amitriptyline, ketotifen, cyclobenzaprine, mianserinand imipramine; anti-platelet drugs, such as aspirin and clopidogrel;anti-coagulants, such as heparin, acenocumarol, warfarin, dabigatran andrivaroxaban; and antioxidants, such as edaravone; the tissue plasminogenactivator and the recombinant variants thereof; nucleic acids which havethe capability of silencing the expression of genes involved in apathology characterized by an increase in expression of TLR-4 and/or anincrease in activation of TLR-4, such as antisense RNA, antisense DNAand small interfering RNA; peptides, such as signaling peptides andtarget-binding peptides.

Pharmaceutical Compositions

For the administration to a subject in need of a nucleic acid aptamerwith the capability of binding specifically to and inhibiting TLR-4 andcomprising a sequence selected from the group consisting of SEQ ID NO: 1and SEQ ID NO: 2 or a functionally equivalent variant thereof, or acomplex comprising a nucleic acid aptamer with the capability of bindingspecifically to and inhibiting TLR-4 and comprising a sequence selectedfrom the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2 or afunctionally equivalent variant thereof and a functional group, saidaptamers and complexes can be formulated in suitable pharmaceuticalcompositions.

In another aspect, the present invention relates to a pharmaceuticalcomposition, hereinafter “the first pharmaceutical composition of theinvention”, comprising at least one nucleic acid aptamer with thecapability of binding specifically to and inhibiting TLR-4 andcomprising a sequence selected from the group consisting of SEQ ID NO: 1and SEQ ID NO: 2 or a functionally equivalent variant thereof.

In a particular embodiment, the first pharmaceutical composition of theinvention further comprises one or more pharmaceutically acceptablecarriers, excipients, or solvents.

In another aspect, the present invention relates to a pharmaceuticalcomposition, hereinafter “the second pharmaceutical composition of theinvention”, comprising at least one complex comprising a nucleic acidaptamer with the capability of binding specifically to and inhibitingTLR-4 and comprising a sequence selected from the group consisting ofSEQ ID NO: 1 and SEQ ID NO: 2 or a functionally equivalent variantthereof and a functional group.

In a particular embodiment, the second pharmaceutical composition of theinvention further comprises one or more pharmaceutically acceptablecarriers, excipients, or solvents.

The pharmaceutical compositions provided by the present invention can beadministered to a subject for the treatment of a pathology characterizedby an increase in expression of TLR-4 and/or an increase in activationof TLR-4.

The terms “aptamer”, “TLR-4”, “functionally equivalent variant”,“complex”, “functional group”, “drug”, “treatment”, “subject” and“pathology characterized by an increase in expression of TLR-4 and/or anincrease in activation of TLR-4” have been described in detail above andtheir definitions and particularities are herein included by reference.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” or “pharmaceutically acceptable solvent”, in thecontext of the present invention, seeks to include any and all of thesolvents, dispersion media, coatings, antibacterial and antifungalagents, absorption delaying and isotonic agents, and the like,compatible with the pharmaceutical administration. The use of suchcarriers and vehicles in pharmaceutically active substances iswell-known in the art. Unless any conventional carrier is incompatiblewith the active compound, use thereof in the compositions of theinvention is contemplated. The acceptable vehicles, excipients, oracceptable stabilizers are not toxic for the subject at the doses andconcentrations used, and include buffers such as phosphate, citrate, andother organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride, hexamethonium chloride, benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol, andm-cresol); low molecular weight polypeptides (less than about 10 aminoacids); proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounterions such as sodium; metal complexes (for example, Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

Supplementary active compounds can also be incorporated in thepharmaceutical composition provided by the present invention. Therefore,in a particular embodiment, the pharmaceutical composition provided bythe present invention can also contain more than one active compound asrequired for the particular indication in question, preferably thosewith complementary activities that do not adversely affect one another.For example, it may desirable to furthermore provide a chemotherapeuticagent, a cytokine, an analgesic agent, an anti-inflammatory agent or animmunosuppressive agent. The effective amount of said other activeagents depends, among other things, on the therapeutic amount of theaptamers or of the complexes which are present in the pharmaceuticalcomposition, the nature and the seriousness of the pathology to betreated, the subject, etc.

In one embodiment, the nucleic acid aptamer with the capability ofbinding specifically to and inhibiting TLR-4 and comprising a sequenceselected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2 or afunctionally equivalent variant thereof, or the complex of theinvention, are formulated with vehicles that will protect said productsfrom rapid elimination from the body, such as a controlled releaseformulation, including implants and microencapsulated delivery system.Biodegradable and biocompatible polymers, such as ethylene-vinylacetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters,and polylactic acid can be used. The methods for the preparation of suchformulations will be evident for persons skilled in the art. These canbe prepared according to methods known by persons skilled in the art,for example, as described in U.S. Pat. No. 4,522,811.

The pharmaceutical compositions provided by the present invention can beadministered to a subject by means of any suitable administration route,such as, for example, by parenteral route.

The term “parenteral”, in the context of the present invention, includesthe intravenous, intraperitoneal, intramuscular, or subcutaneousadministration. The intravenous form of parenteral administration isgenerally preferred.

Furthermore, the pharmaceutical compositions provided by the presentinvention can be suitably administered by pulse infusion, for example,with decreasing does of the aptamer or of the complex of the invention.Preferably, the dosage is provided by means of injections, morepreferably intravenous or subcutaneous injections, depending in part onwhether the administration is brief or chronic.

In another particular embodiment, the pharmaceutical compositionsprovided by the present invention can be adapted for parenteraladministration with the addition of sterile solutions, suspensions orlyophilized products in the suitable dosage form. The pharmaceuticalcompositions suitable for injectable use include sterile aqueoussolutions or dispersions or sterile powders for the preparation ofsterile injectable solutions or dispersions. For intravenousadministration, suitable vehicles include physiological saline solution,bacteriostatic water CremophorEM (BASF, Parsippany, N.J.) or phosphatebuffer saline (PBS). In all cases, the composition must be sterile andfluid to facilitate injectability. It must be stable in themanufacturing and storage conditions and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thevehicle can be a solvent or dispersion medium which contains, forexample, water, ethanol, a pharmaceutically acceptable polyol such asglycerol, propylene glycol, liquid polyethylene glycol, and suitablemixtures thereof. Suitable fluidity can be maintained, for example, bymeans of the use of a coating such as lecithine, by means of maintainingthe particle size required in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can beachieved by means of various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include in thecomposition isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, and sodium chloride. The prolonged absorption of theinjectable compositions can be provoked, including in the composition anabsorption-delaying agent, for example, aluminum monostearate and/orgelatin.

The injectable sterile solutions can be prepared by incorporating therequired amount of the active compound (for example, an aptamer orcomplex of the invention) in a suitable solvent with one or acombination of the previously listed ingredients, as required, followedby filtration sterilization. Generally, the dispersions are prepared byincorporating the active compound in a sterile vehicle which contains abasic dispersion medium and the other required ingredients from thosepreviously listed. In the case of sterile powders for the preparation ofinjectable sterile solutions, the preferred methods of preparation arevacuum-drying and freeze-drying, which produces a powder of the activeingredient plus any additional desired ingredient from previouslyfiltered sterile solution thereof.

In a particular embodiment, said pharmaceutical composition isadministered through intravenous route. Suitable excipients, such asbulking agents, buffering agents or surfactants, can be used. Thementioned formulations will be prepared using standard methods such asthose described or contemplated in the Spanish and United Statespharmacopoeias and similar reference texts.

It is particularly advantageous to formulate the pharmaceuticalcompositions, namely, the parenteral compositions, in the dosage unitform to facilitate the dosage administration and uniformity. Dosage unitform, as it is used herein, refers to physically discrete units suitableas unit dosages for the subject to be treated, each unit containing apredetermined amount of active compound (an aptamer or complex of theinvention) calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical vehicle. The specificationfor the unit dosage forms of the invention are conditioned by and dependdirectly on the unique features of the active compound and theparticular therapeutic effect to be achieved, and the inherentlimitations in the composition technique of such active compound for thetreatment of subjects.

The active compounds (aptamer or complex of the invention) willtypically be administered one or more times a day, for example 1, 2, 3or 4 times a day, with typical total daily doses in the interval of0.0001 to 1.000 mg/kg of body weight/day, preferably from about 0.001 toabout 100 mg/kg of body weight/day, more preferably from about 0.01 to10 mg/kg of body weight/day. The pharmaceutical compositions can beformulated for the purpose of containing the desired amount, such as atherapeutically effective amount of the aptamer or complex of theinvention.

The pharmaceutical compositions provided by the present invention can beincluded in a container, packaging, or dispenser together withinstructions for administration.

Imaging Methods of the Invention

In another aspect, the invention relates to the use of a complexaccording to the invention for in vivo imaging of a cell, tissue ororgan which express TLR4, wherein said complex comprises one or moreaptamers according to the invention and a functional group, saidfunctional group being a detectable moiety.

Suitable detectable moieties for use in the in vivo imaging methodsaccording to the invention have been described above in the context ofthe complex of the invention and include, without limitation, aradionuclide, a fluorophore, a contrast media, a protein and an haptene.

The invention is described below by means of the following exampleswhich are merely illustrative and by no means limiting of the scope ofthe invention.

EXAMPLES Materials and Methods Aptamer Library

The inventors used the RND40 aptamer library to carry out the screeningof aptamers specific for TLR-4, supplied by IBA GmbH (Goettingen,Germany). The initial RND40 library is theoretically made up of 1024single-stranded DNA (ssDNA) oligonucleotides with fixed sequence at theends, consisting of 18 nucleotides where each hybridize the respectiveprimers for PCR amplification thereof, and a central region consistingof 40 bases having random sequence. In the screenings made, 1013oligonucleotides from this library have been used.

6HIS-Recombinant hTLR-4

The protein corresponding to the extracellular domain of the human TLR-4protein, amino acids 24-631, was recombinantly generated fused to a6-histidine tag at the C-terminal end, by means of expression inbaculovirus.

Cells

HEK293T and HEK293T/TLR-4 cells were obtained from Invivogen (San Diego,Calif., USA).

Screening with HEK-293T-TLR4/HEK-293T Cells

For each screening round, 8-10×10⁵ HEK-293T cells were seeded intriplicate in P6 plate wells, 24 h before the screening assay and wereincubated at 37° C., 5% CO₂. Then 1 nmol of aptamers from the RND40library (or from the population isolated in the preceding screeninground) in 100 μl PBS, which aptamers were previously denatured at 95° C.for 10 min followed by incubation at 4° C. for 10 min, was added; 300 μlof DMEM medium (Dulbecco's modified Eagle's medium) supplemented with10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and25 μg/ml amphotericin were added and applied on the cells. After 1 h ofincubation at 37° C., 5% CO₂, the culture medium with the non-boundaptamers was removed, the cells were washed twice with PBS and recoveredin 500 μl of PBS by means of centrifugation at rpm. The cells werecentrifuged to remove the supernatant and the aptamers adhered to thecells were amplified by PCR to prepare a sufficient amount for thefollowing screening round.

The counter-screening on HEK-293T cells from the RND40 aptamer librarywas done during the prior preparation of the initial RND40 populationand every 3 screening rounds, with the population isolated from thepreceding screening round. To that end, 8-10×10⁵ HEK-293T cells wereseeded in triplicate in P6 plate wells, 24 h before the screening assayand were incubated at 37° C., 5% CO₂. Then, 1 nmol of aptamers of theRND40 library (or of the population isolated in the preceding screeninground) in μl PBS, which aptamers were previously denatured at 95° C. for10 min followed by incubation at 4° C. for 10 min, was added; 300 μl ofDMEM medium (Dulbecco's modified Eagle's medium) supplemented with 10%fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 25μg/ml amphotericin were added and applied on the cells. After 1 h ofincubation at 37° C., 5% CO₂, the culture medium with the non-boundaptamers for being used in screening rounds on TLR-4 was removed.

Screening with Soluble hTLR-4 Protein

For each screening round, the RND40 library enriched in a precedingscreening round is used. 1 nmol of aptamers was incubated with 7 μg of6×HIS-hTLR-4 (at a ratio of 10 aptamer molecules to 1 hTLR-4 molecule),in an aptamer buffer (20 mM Tris-HCl, pH 7.4, 1 mM MgCl₂, 150 mM NaCl, 5mM KCl), at 37° C. for 1 h with stirring. Subsequently, NTA-Ni resin(QIAGEN, Germany) was added and it was incubated at 4° C. for 1 h tocapture the protein. After 3 washes with aptamer buffer, the boundsequences were amplified by PCR to prepare a sufficient amount for thefollowing screening round.

The counter-screening is performed in the same conditions as screeningbut in the absence of TLR-4 protein bound to the resin.

Amplification of the Selected Aptamers

The selected aptamers were resuspended in a volume of 20 μl of distilledwater and amplified by means of PCR using the primers, which willcorrespond with sequences SEQ ID NO: 5 (GCGGATGAAGACTGGTGT) and SEQ IDNO: 6 (GTTGCTCGTATTTAGGGC) in the conditions of 0.8 μM/primer SEQ ID NO:5, 0.8 μM/primer SEQ ID NO: 6, 200 mM dNTPs, 2 mM MgCl₂, 10 U Taqpolymerase (Biotools, Spain) in a final volume of 200 μl according tothe following amplification program: 2 min at 95° C.; 15 cycles of 30 sat 95° C., 30 s at 56° C. and 30 s at 72° C.; and finally 5 min at 72°C.

ELONA

It was determined if the selected aptamers recognized the TLR-4 protein.To that end, 100 ng/well of 6×HIS-recombinant TLR-4 were added to a96-well microtiter plate and were incubated at 4° C. for 16 h.Subsequently, individual aptamers labeled with digoxigenin in the 5′ endwere diluted at a concentration of 5 μg/mL and then denatured for 10 minat 95° C. and cooled for 10 min on ice. Then, 20 pmol of each of theaptamers in 100 μl (200 nM) of aptamer buffer were added to each welland the plate was incubated for 1 h at 37° C. Finally, the plate wasincubated with peroxidase-conjugated anti-digoxigenin antibodies anddeveloped using ABTS. An anti-Li H2A DNA aptamer was used as a positivecontrol (Martin et al., 2013, PLoS ONE 8: e78886).

Binding Assays for Binding the Aptamers to Recombinant hTLR-4

For the purpose of analyzing the capability of each of the identifiedaptamers of binding to hTLR-4, experiments were performed in whichhTLR-4 was bound to a Ni-NTA resin, previously equilibrated, through thehistidine tag. Then, the resin:protein complexes containing 1 pmol ofTLR4 were incubated with 1 pmol of aptamers TLRApt #1R (SEQ ID NO: 3),TLRApt #2F, TLRApt #3R and TLRApt #4F (SEQ ID NO: 4), previouslystructured by means of thermal denaturation at 95° C. for 10 min andsubsequent renaturation at 4° C. for 10 min. After incubating for 10 minat 37° C., the complexes were washed and the aptamers were recoveredwith 150 mM imidazol dissolved in PBS with 1 mM MgCl₂. The values ofaptamer bound to hTLR-4 were determined by means of quantitative PCR(qPCR) using the primers with sequences SEQ ID NO: 5 and SEQ ID NO: 6 inan IQ5 thermal cycler (BioRad).

Binding Assays for Binding the Aptamers to TLR-4 Expressed in Cells

For the purpose of analyzing the capability of the identified aptamersof binding to the TLR-4 protein expressed in HEK-293 cells, 20 pmol ofeach of the aptamers TLRApt #1R (SEQ ID NO: 3), TLRApt #2F, TLRApt #3Rand TLRApt #4F (SEQ ID NO: 4) were added to a HEK-293-TLR4 cell cultureseeded at 20,000 cells/well in 96-well microtiter plates at a density of2×10⁴ cells/well, 2 days before the start of the assay. After incubatingfor 30 min at 37° C., 5% CO₂, the cells were washed, the aptamers wererecovered with 150 mM imidazol dissolved in PBS with 1 mM MgCl₂, andqPCR was carried out to determine the values of Ct.

Receptor hTLR-4 Activity Assays

To perform these assays, HEK-Blue hTLR4 cells (Invivogen, ref.hkb-htlr4), expressing human receptor TLR-4, together with the MD2 andCD14 proteins, which are activated by the binding of their agonist,lipopolysaccharide from Escherichia coli K12 (LPS-EK), were used. Fordetecting activation of TLR-4, this cell line contains a SEAP (secretedembryonic alkaline phosphatase) reporter gene, which is controlled bythe NF-kB promoter, such that it is expressed in response to this NF-kBsignaling route, induced by TLR-4. The SEAP enzyme is secreted into theculture medium, and by adding and metabolizing its commercial substrateQUANTI-Blue™ (Invivogen, San Diego, Calif., USA) it causes a change incolor of the medium from red to blue. In addition, the control agonistmolecule LPS-EK UP (lipopolysaccharide from E. coli K12, Ultra Pure) andantagonist LPS-RS UP (lipopolysaccharide from R. sphaeroides, UltraPure) are dissolved in 1 mM MgCl₂ in sterile PBS at concentrations of0.02 ng/μL and 2 ng/μL, respectively. The aptamers are prepared atconcentrations of 0.1, 1, 10 and 100 ng/μL in 1 mM Cl₂Mg in sterile PBS,are denatured at 95° C. for 10 min and were structured at 4° C. for 10min.

For the assay, HEK Blue-hTLR4 cells were seeded onto 96-well cultureplates at 2×10⁴ cells/well in 200 μL complete medium DMEM supplementedwith (1×) HEK-Blue™ Selection. After 24 h or 48 h incubation, when thecells get 70-80% confluence, the medium is recovered and added 170 μLfresh medium. In control wells 30 μL SELEX buffer is added. In the otherwells, 20 μL of LPS-EK-ultra pure (Invivogen, USA) at 20 ng/mL (0.1ng/mL final) or 20 μL of lysate from 1.5-2.5×107 HEK293 cells/mL(Damage-associated molecular pattern; DAMP) were added as agonistmolecules. After 1 h incubation, 10 μL aptamer diluted in SELEX bufferto the appropriate concentrations were added to the wells to reach finalconcentrations indicated in the figures. LPS-RS ultra-pure (Invivogen,USA) at a concentration of 200 ng/mL was used as antagonist control.Secreted embryonic alkaline phosphatase (SEAP) activity was measuredafter 24 h using QUANTI-Blue™ substrate (Invivogen) at 630 nm.

Effect of Aptamers on Macrophages

Peritoneal macrophages were seeded in 12-well plates at a density of1×10⁶ cells/ml. Macrophages were stimulated in the presence of 500 ng/mlLPS and 1 h after the aptamer was added to a final concentration of 20nM and 200 nM. Nitrites release was measured by the Griess reactionafter 24 h. Samples were assayed in duplicate.

Animal Model of Stroke

Adult male C57BL mice weighing 28 to 30 g were used. C57BL/10ScNJ(formerly called C57BL/10ScCr) and C57BL/10ScSn mice were acquired fromThe Jackson Laboratory (Bar Harbor, Me., USA). The murine strainC57BL/10ScNJ does not express functional TLR4 by deletion of the TLR4gene, and the C57BL/10ScSn strain does not express any mutation in theTLR4 gene and is used as a control group. 4 TLR4-deficient mice wereused per group (C57BL/10ScNJ) and 4 control mice were used per group(C57BL/10ScSn). All the experimental protocols complied with theguidelines of the “Comite de Bienestar Animal” (Animal Well-beingCommittee) of the Universidad Complutense (following European Directives86/609/EEC and 32/2007/EC). The animals were housed in normaltemperature conditions, moisture conditions and 12-hour light/darknesscycle conditions with free access to food and water.

The induction of focal cerebral ischemia was carried out by means ofmedian cerebral arterial occlusion (MCAO) according to the teachings ofCaso et al., 2007 (Caso et al., 2007, Circulation 115:1599-608).Briefly, permanent focal cerebral ischemia is induced by ligature of thecommon carotid artery (CCA) and distal ipsilateral occlusion of themedian cerebral artery (MCA). For the ligature of the CCA, aventral-cervical incision is made to isolate said artery and occlude itpermanently via ligature. For occlusion of the MCA, an incision is madeat 1 cm of the perpendicular line joining the lateral cantus of the lefteye and the outer ear canal and the temporal muscle is removed. A drillhole is made to expose the MCA that is occluded by ligature. Aftersurgery, closure of the incisions and disinfection, the animals arereturned to their cages with free access to water and food. The animal'svital signs are controlled during surgery.

Brain damage was evaluated by means of magnetic resonance imaging.Briefly, the mice were anesthetized with isoflurane and 24 hours afterMCAO, the size of the infarction was evaluated by MRI. The imageshighlighted in T2 (T2WI) were acquired in a BIOSPEC BMT 47/40 operatingat 4.7 T (Bruker-Medical, Ettlingen, Germany; MRI Unit, InstitutoPluridisciplinar, UCM).

Flow Cytometry Analysis

All flow cytometry analyses were performed on a FACScan model flowcytometer (Becton Dickinson Immunocytometry systems). Binding ofaptamers to cell surface TLR4 was analyzed by seeding HEK293 or HEKBlue-hTLR4 cells onto 24-well culture plates at 2×10⁵ cells/well in 200μL complete medium DMEM supplemented with HEK-Blue™ Selection buffer.Afterwards, cells are treated or not with the TLR-4 activator LPS-EK-UP(0.4 ng/well) for 30 min and then with the Alexa Fluor 488-labelledaptamers (20 nM) in 50 μL volume of PBS buffer containing 1 mM MgCl₂ and1 mg/ml BSA for 30 min at room temperature in the dark. Cells were thenwashed with 2 mL of the same buffer, suspended in 0.5 mL of the bufferand subjected to flow cytometry analysis.

Nucleases Digestion

Three hundred ng of aptamers were folded in SELEX buffer by heating to95° C. and cooling on ice. Refolded aptamer were incubated with 2 U of AExonuclease or DNAse I (Fermentas) in a 10 μL reaction for 10 min, 30min, 1 h, 2 h and 4 h at 37° C. Afterward, samples were solved on a 3%agarose gel. Bands were visualized by GelRed (Biotium) and quantifiedusing Image Studio Digits V3.1 software.

Example 1: Screening of Aptamers Specific for TLR-4

The inventors used the RND40 aptamer library to carry out the screeningof aptamers specific for TLR-4 supplied by IBA GmbH (Goettingen,Germany). The initial RND40 library is made up of oligonucleotides(ssDNA) with fixed sequence at the ends, consisting of 18 nucleotideseach, where they hybridize the respective primers for PCR amplificationthereof, and a central region consisting of 40 bases having a randomsequence.

The initial RND40 library of 1013 aptamers was enriched in aptamersspecific for hTLR-4. As a prior step to screening with TLR-4, a“counter-screening” process was performed on the library for the surfaceor matrix where the target molecule (magnetic resin, cells of the sameline as that which expresses the target protein, etc.) is presented.

Example 2: Characterization of the Selected Aptamers

The selected aptamers were identified after 6 screening rounds accordingto the following strategies:

-   -   a) By means of cloning of the population of aptamers into a        plasmid for the purpose of obtaining individual aptamers, and        subsequent Sanger sequencing.    -   b) By means of massive sequencing of the population of aptamers        obtained the aptamers that are the most repeated being        identified.

The sequences that are most represented were chemically synthesized byIBA GmbH (Goettingen, Germany) and the affinity and activity of each ofthe aptamers was studied in binding assays for binding to recombinanthTLR-4 protein or to HEK-293 cells-TLR-4, by means of ELONA, bindingassays for the binding of the aptamers to the recombinant TLR-4 proteinand to TLR-4 expressed in cells, receptor hTLR-4 activity assays.

The results of the ELONA assays (FIG. 1 ) clearly show that the aptamersthat more efficiently bind to the recombinant hTLR-4 protein areaptamers TLRApt #1R (SEQ ID NO: 3) and TLRApt #4F (SEQ ID NO: 4). Bymeans of the same type of assay, aptamers TLRApt #2F and TLRApt #3R wereidentified. FIG. 2 shows the most likely sequences and secondarystructures of the selected aptamers TLRApt #1R (SEQ ID NO: 3) and TLRApt#4F (SEQ ID NO: 4), obtained using mFold software (Zuker M., 2003,Nucleic Acids Res 31:3406-15).

The capability of binding of the aptamers to hTLR-4 was determined bymeans of incubation of the aptamers with a Ni-NTA resin with boundhTLR-4, recovery and subsequent qPCR amplification of the boundaptamers. The obtained results show that all the selected aptamers areable to bind to the recombinant hTLR-4 protein (FIG. 3A). In theseexperiments, a lower Ct value indicates a larger amount of aptamer boundto hTLR-4.

For the purpose of analyzing the capability of the identified aptamersof binding to the TLR-4 protein expressed in HEK-293 cells, 20 pmol (500ng) of aptamers TLRApt #1R (SEQ ID NO: 3) and TLRApt #4F (SEQ ID NO: 4)are added to a HEK-293-TLR4 cell culture. After incubating for 30 min at37° C., 5% CO₂, the cells are washed and recovered, and qPCR isperformed for the purpose of calculating the Ct values. In theseexperiments, a lower Ct value indicates a larger amount of aptamer boundto the cells. The obtained results show that the selected aptamersTLRApt #1R (SEQ ID NO: 3) and TLRApt #4F (SEQ ID NO: 4) (FIG. 3B) arethose which bind with a higher affinity.

Dose-response curves were made for the purpose of determining theconcentration of each aptamer at which the maximum antagonist effect isobtained using as agonist LPS-EK-UP (FIG. 4A) or lysates from HEK293cells (Damage-associated molecular pattern; DAMP) (FIG. 4B). Based onthe results obtained, it can be concluded that the concentrations atwhich the best effect is observed are 20 nM for agonist LPS-EK-UP and200 nM for DAMPs.

The effect of the aptamers on macrophages is shown in FIG. 5 . AptamersTLRApt #4F (SEQ ID NO: 4) and TLRApt #4F-T (SEQ ID NO: 2) inhibitednitrite release after stimulation of macrophages with LPS. In theseexperiments, aptamer TLRApt #4F (SEQ ID NO: 4) seems more active thanTLRApt #4F-T (SEQ ID NO: 2) for the same concentrations.

Example 3: Optimization of the Aptamer Antagonists of TLR-4 forIncreasing its Activity and Stability in Animal Models

The aptamers that showed the capability of inhibiting receptor TLR-4have been modified by means of removing specific regions from thesequence thereof for the purpose of increasing the stability and/orresistance with respect to nucleases. To that end, a study of thesecondary structure of the different aptamers was conducted using themFold program (Zuker M., 2003, mentioned at supra) and the capability ofthe aptamers of forming G-quadruplex structures has been analyzed bymeans of the QGRS Mapper program (Kikin et al., 2006, Nucleic Acids Res34:W676-W682). Therefore, aptamers TLRApt #1R-T (SEQ ID NO: 1) andTLRApt #4F-T (SEQ ID NO: 2), corresponding to those identified in thefirst screenings (FIG. 6 ), were designed and synthesized.

Example 4: Effect of the Aptamer Antagonists of TLR-4 in an Animal Modelof Stroke

The capability of the new aptamers, optimized for blocking theinflammatory response produced after an episode of stroke in an animalmodel of stroke, evaluating the capability of the aptamers specific forTLR-4 of reducing the cerebral injury.

To that end, adult male TLR-4-deficient mice (C57BL/10ScNJ) and controlmice expressing TLR-4 (C57BL/10ScSn) were used, in which focal cerebralischemia was induced by means of median cerebral arterial occlusion(MCAO). The results obtained in mice normally expressing TLR4 (controlmice) demonstrate that the aptamers cause a reduction in size of theinfarcted area which, in the case of aptamer TLRApt #4F-T (SEQ ID NO:2), is statistically significant. In addition, the results obtained inTLR-4-deficient mice show that there is no effect of aptamer TLRApt#4F-T (SEQ ID NO: 2) on which it is obtained when the mice are treatedwith the vehicle (FIG. 7 ). This data clearly indicates that the effectof aptamer TLRApt #4F-T (SEQ ID NO: 2) occurs through TLR-4.

In another set of experiments, adult male mice C57BL/10ScSn (WT; normal)were subjected to induction of a focal cerebral ischemia and thentreated with intraperitoneal injection of different amounts of aptamersTLRApt #4F-T (SEQ ID NO: 2) and TLRApt #4F (SEQ ID NO: 4) or vehicle(PBS+1 mM Mg²⁺). The results obtained demonstrate that the aptamerscause the higher reduction in size of the infarcted area at 1 nmolaptamer/animal and that this reduction is statistically significant(FIG. 8 ).

Example 5: Flow Cytometry

Flow cytometry assays were performed using HEK293 cell line transfectedwith human TLR4, 293-hTLR4A. The parental human HEK293 cell line,lacking human TLR4, was used as a control. In this experiment aptamerswere labeled with Alexa Fluor 488. FIG. 9A shows that Alexa Fluor488-labelled aptamers bind strongly to 293-hTLR4A cells (right panel),but not to HK293 cells lacking human TLR4 (left panel). In addition, itis observed that ApTLR #4F-T (SEQ ID NO: 2) binds to the target withhigher affinity than ApTLR #1R-T (SEQ ID NO: 1). In turn, the results ofcellular staining using the selected aptamers after activation (or not)of hTLR4-Blue-HEK cells were compared. As expected, aptamers bound TLR4after activation in a similar level relative to non-activated cells(FIG. 9B).

Example 6: Half-Life Calculation by Nucleases Digestion

The half-life of the aptamers was measured in vitro in the presence of AExonuclease or DNAse I (FIG. 10 ). Results show that the four aptamersare resistant to λ Exonuclease meanwhile DNAse I produces atime-dependent degradation of the four aptamers. Thus, aptamer ApTLR#1R-T (SEQ ID NO: 1) is the most sensible and is completely degradedafter 5 min incubation in presence of DNAse I. On the contrary, aptamersApTLR #4F (SEQ ID NO: 4) and ApTLR #4F-T (SEQ ID NO: 2) are resistanteven after 2 h incubation with DNAse I.

CONCLUSIONS

-   -   Aptamers TLRApt #1R (SEQ ID NO: 3) and TLRApt #4F (SEQ ID NO: 4)        have been selected with respect to the extracellular domain of        receptor TLR-4, which recognize human receptor TLR-4, both in        its soluble recombinant form (in vitro) and integrated in the        membrane of HEK293 cells (in vivo).    -   Aptamers TLRApt #1R (SEQ ID NO: 3) and TLRApt #4F (SEQ ID NO: 4)        show an antagonist effect of receptor TLR-4.    -   Aptamers TLRApt #1R-T (SEQ ID NO: 1) and TLRApt #4F-T (SEQ ID        NO: 2) have been obtained in truncated form maintaining the        antagonist activity of the original aptamers TLRApt #1R (SEQ ID        NO: 3) and TLRApt #4F (SEQ ID NO: 4), respectively.    -   Aptamer TLRApt #4F-T (SEQ ID NO: 2) is able to reduce the        infarcted area in an animal model of stroke.    -   Aptamers TLRApt #1R (SEQ ID NO: 3) y TLRApt #4F (SEQ ID NO: 4),        selected against the extracellular domain of TLR4 receptor,        recognize the human TLR-4 receptor in both its recombinant        soluble form (in vitro), as integrated in the membrane of HEK293        cells (in vivo).

1-25. (canceled)
 26. A method to improve one or more symptoms of thepathology of sepsis in a subject in need thereof comprisingadministering to the subject an effective amount of an active compoundcomprising at least one nucleic acid aptamer or chemically modifiedvariant thereof, wherein said at least one nucleic acid aptamer orchemically modified variant thereof specifically binds to at least onetarget site on the extracellular domain of human TLR-4 (Toll-likereceptor 4) on at least one target cell, wherein (i) the specificbinding of the at least one nucleic acid aptamer or chemically modifiedvariant thereof to the extracellular domain of human TLR-4 results in adecrease in TLR-4 activity; and (ii) the decrease in TLR-4 activityimproves one or more symptoms of the pathology of sepsis in the subject,wherein the at least one nucleic acid aptamer or chemically modifiedvariant thereof comprises a sequence at least 70% identical to SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, or a combinationthereof.
 27. The method of claim 26, wherein the at least one nucleicacid aptamer or chemically modified variant thereof comprises (a) 1, 2,3, 4, 5, 10, 15, or 20 additional nucleotides at the 5′ end of thesequence of SEQ ID NO: 1 or SEQ ID NO: 2; (b) 1, 2, 3, 4, 5, 10, 15, or20 additional nucleotides at the 3′ end of the sequence of SEQ ID NO: 1or SEQ ID NO: 2; or, (c) a combination thereof.
 28. The method of claim26, wherein the at least one nucleic acid aptamer sequence is selectedfrom the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3and SEQ ID NO: 4, or a combination thereof.
 29. The method of claim 26,wherein the at least one target site can specifically bind to at leastone nucleic acid aptamer of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 orSEQ ID NO:4, or a combination thereof.
 30. The method of claim 26,wherein the subject is a human.
 31. The method of claim 26, wherein theactive compound comprises a complex.
 32. The method of claim 26, whereinthe active compound comprises a conjugate.
 33. The method of claim 26,wherein the active compound further comprises a functional groupcomprising a drug, a nanoparticle, or a combination thereof.
 34. Themethod of claim 33, wherein the drug comprises a TLR-4 antagonist, ananti-inflammatory agent, a nucleic acid, a peptide, or a combinationthereof.
 35. The method of claim 33, wherein the nanoparticle comprisesa polymeric nanoparticle, a lipid nanoparticle, a metal nanoparticle, ora combination thereof.
 36. The method of claim 35, wherein the metal isselected from the group consisting of gold, silver, copper, aluminum,platinum, iron, cobalt, palladium, and combinations thereof.
 37. Themethod of claim 26, wherein the at least one nucleic acid aptamer orchemically modified variant thereof comprises at least one nucleic acidanalogue.
 38. The method of claim 26, wherein the decrease in TLR-4activity results in a reduction and/or interruption in the release of atleast one pro-inflammatory cytokine.
 39. The method of claim 38, whereinthe at least one pro-inflammatory cytokine is selected from the groupconsisting of IL-1, IL-8, TNF-alpha, IL-12, and combinations thereof.40. The method of claim 26, wherein the one or more symptoms of thepathology of sepsis comprises fever, hyperthermia, tachypnea,tachycardia, leukocytosis, or a combination thereof.
 41. The method ofclaim 26, wherein the method further comprises the administration of ananti-inflammatory agent or an anti-coagulant.
 42. The method of claim26, wherein the sepsis is caused by a pathogenic microorganism.
 43. Apharmaceutical composition for the treatment of sepsis comprising: (a) atherapeutically effective amount of an active compound comprising atleast one nucleic acid aptamer or chemically modified variant thereof,wherein said at least one nucleic acid aptamer or chemically modifiedvariant thereof specifically binds to at least one target site on theextracellular domain of human TLR-4 (Toll-like receptor 4) on at leastone target cell, wherein (i) the specific binding of the at least onenucleic acid aptamer or chemically modified variant thereof to theextracellular domain of human TLR-4 results in a decrease in TLR-4activity; and (ii) the decrease in TLR-4 activity improves one or moresymptoms of the pathology of sepsis in the subject, wherein the at leastone nucleic acid aptamer or chemically modified variant thereofcomprises a sequence at least 70% identical to SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3 or SEQ ID NO: 4, or a combination thereof; and, (b) atleast one additional therapeutic agent for the treatment of sepsis. 44.The pharmaceutical composition of claim 43, wherein the at least oneadditional therapeutic agent for the treatment of sepsis comprises aTLR-4 antagonist agent, an anti-inflammatory agent, an anticoagulant, anantioxidant, an analgesic agent, an immunosuppressive agent, or acytokine.
 45. A reagent for the in vitro detection of TLR-4 in abiological sample comprising at least one nucleic acid aptamer orchemically modified variant thereof, wherein said at least one nucleicacid aptamer or chemically modified variant thereof specifically bindsto at least one target site on the extracellular domain of human TLR-4on at least one target cell, wherein the specific binding of the atleast one nucleic acid aptamer or chemically modified variant thereof tothe extracellular domain of human TLR-4 results in a decrease in TLR-4activity; and wherein the at least one nucleic acid aptamer orchemically modified variant thereof comprises a sequence at least 70%identical to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4,or a combination thereof.